Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of Ca_V3.1 T-type Ca²⁺ channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking Ca_V3.1 channels (Ca_V3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that Ca_V3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (Ca_V3.1 WT). Intriguingly, we also found that Ca_V3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, Ca_V3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in Ca_V3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of Ca_V3.1 WT mice, during unconsciousness. These results suggest that the Ca_V3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.Thalamocortical interactive rhythmic activities are well-defined physiological correlates of both conscious and unconscious conditions (1, 2). From a functional perspective, abnormal slow cortical rhythms and their synchronized network dynamics are omnipresent correlates of unconscious states, such as coma and general anesthesia (3, 4). Moreover, a dynamic alteration of coherence as well as coupling/uncoupling in thalamocortical circuits also can be characterized as likely correlates of unconsciousness (3–5).Since the discovery of low threshold, T-type Ca²⁺ channels (6, 7) and the subsequent studies of intrinsic electrophysiological properties in the thalamic neurons (8, 9), T-type Ca²⁺ channels have been implicated in many physiological and pathological brain states (for a review, see ref. 10). The ionic conductances they support have been shown to generate synchronized oscillatory activity in thalamocortical circuits through calcium-dependent low-threshold spikes (LTSs). Indeed, these LTSs, generated by “deinactivation” of T-type Ca²⁺ channels, underlie thalamic burst firing. This activity is reflected as high-amplitude low-frequency oscillations in electroencephalography, and its presence is recognized as spike-wave-discharges, low-frequency rhythms (<1 Hz slow, delta and theta rhythms), as well as by spindle-generated rhythmicity (10).Recent molecular genetic studies coupled with electrophysiological and behavioral approaches confirmed the classical view that Ca_V3.1 channels play a central role in the generation of thalamocortical rhythms, such as 3- to 4-Hz spike-wave discharge during absence seizures (11, 12). Regarding the role of Ca_V3.1 in slow wave sleep, however, mice with such genetic deletions present electrophysiological consequences that are inconsistent with the above generalization, even with behavioral phenotypes exhibiting fragmented sleep. Indeed, mice with a global Ca_V3.1 deletion showed reduced delta rhythm (13) in contrast to the increased delta rhythms found in mice with thalamus-restricted deletion of Ca_V3.1 (14). In addition, there is clear evidence that thalamic T-type Ca²⁺ channels support the generation of spindle oscillations (15). However, recently published work proposes that sleep spindles are sustained in mice lacking Ca_V3.1 channels (16). These results differ from the classical view and raise the need to examine further the role of thalamic Ca_V3.1 channels in the generation of thalamocortical rhythms.Here we addressed the issue of whether Ca_V3.1 channels are important for the generation of low-frequency thalamocortical rhythms during unconsciousness. Spectral analysis of EEG recordings from Ca_V3.1 KO mice indicates a shift away from low frequency, with an increase probability toward the high-frequency rhythmic components. There is also a significant alteration of thalamocortical dynamic interactions.     

Rebound burst firing in the reticular thalamus is not essential for pharmacological absence seizures in mice

Intrinsic burst and rhythmic burst discharges (RBDs) are elicited by activation of T-type Ca²⁺ channels in the thalamic reticular nucleus (TRN). TRN bursts are believed to be critical for generation and maintenance of thalamocortical oscillations, leading to the spike-and-wave discharges (SWDs), which are the hallmarks of absence seizures. We observed that the RBDs were completely abolished, whereas tonic firing was significantly increased, in TRN neurons from mice in which the gene for the T-type Ca²⁺ channel, Ca_V3.3, was deleted (Ca_V3.3^−/−). Contrary to expectations, there was an increased susceptibility to drug-induced SWDs both in Ca_V3.3^−/− mice and in mice in which the Ca_V3.3 gene was silenced predominantly in the TRN. Ca_V3.3^−/− mice also showed enhanced inhibitory synaptic drive onto TC neurons. Finally, a double knockout of both Ca_V3.3 and Ca_V3.2, which showed complete elimination of burst firing and RBDs in TRN neurons, also displayed enhanced drug-induced SWDs and absence seizures. On the other hand, tonic firing in the TRN was increased in these mice, suggesting that increased tonic firing in the TRN may be sufficient for drug-induced SWD generation in the absence of burst firing. These results call into question the role of burst firing in TRN neurons in the genesis of SWDs, calling for a rethinking of the mechanism for absence seizure induction.Absence seizures are generalized, nonconvulsive seizures characterized by the appearance of bilaterally synchronous spike-and-wave discharges (SWDs) on the electroencephalogram (EEG). The frequency of the SWDs is variable among different models and is usually higher (4–12 Hz) in rodents than in humans (3 Hz) (1). SWDs represent synchronized oscillations of the thalamocortical network (2–4), a network that includes neurons of the cerebral cortex, thalamocortical nucleus (TC), and thalamic reticular nucleus (TRN) (5). This thalamocortical circuitry is a key CNS structure for gating the flow of sensory information from the periphery to the cortex (6, 7). Both thalamocortical and corticothalamic connections are mainly glutamatergic (8). The TRN is a shell-like structure that covers most of the rostral, lateral, and ventral parts of the thalamus (5) and is composed exclusively of GABAergic interneurons that provide massive inhibitory input to TC neurons (9). The most distinctive feature of thalamocortical circuitry is its intrinsic ability to generate oscillations via the reciprocal circuits between TC and TRN neurons (10–12).Both TC and TRN neurons are able to generate two distinctive patterns of action potential firing: tonic and burst (13, 14). Burst firing is mediated by low-voltage–activated (LVA) T-type Ca²⁺ channels (15). There are three subtypes of T-type Ca²⁺channels, called Ca_V3.1, Ca_V3.2, and Ca_V3.3, each with distinctive expression patterns and kinetic properties (16). Within the thalamocortical circuit, Ca_V3.1 channels are predominantly expressed in TC neurons, whereas Ca_V3.2 and Ca_V3.3 channels are expressed only in TRN neurons (17). Unlike high-voltage–activated Ca²⁺ channels, T-type Ca²⁺ channels are inactivated at membrane potentials around −60 to −50 mV (15). However, when the membrane potential is hyperpolarized for longer than 100 ms, these channels are de-inactivated and can then initiate a burst of action potentials once the membrane potential is repolarized (18). TC neurons with a relatively depolarized resting membrane potential exhibit a tonic mode of firing associated with conventional, Na/K-dependent action potentials upon receiving excitatory inputs. However, when the neurons are hyperpolarized by inhibitory inputs, high-frequency burst firing can be triggered by Ca_V3.1 channels. TRN neurons, on the other hand, have a relatively hyperpolarized resting potential around −70 mV and most of their T-type Ca²⁺ channels are available for activation. These channels, especially Ca_V3.3, enable TRN neurons to generate rhythmic bursts, even in response to excitatory inputs (12, 19). These oscillatory bursts of action potentials in the TRN provide a barrage of hyperpolarizing inhibitory postsynaptic potentials (IPSPs) to the TC neurons, which in turn respond with action potential bursts due to the activation of Ca_V3.1. These TC bursts provide rhythmic excitatory drive to the cortex, eventually resulting in SWDs.Several models have been proposed to account for the mechanism of SWD generation in the thalamocortical circuit. One well-known model argues that intrinsic cortical oscillations drive the synchronized activity of the entire thalamocortical circuit to cause SWDs (20). The most widely accepted model suggests that rhythmic bursting activity in TRN neurons is a key element for initiation and maintenance of SWDs (4, 10, 12). This model has been indirectly supported by previous observations that lesions or blockade of voltage-gated Ca²⁺ channels in TRN disrupt SWDs (21), leading to the idea that the degree of the synchrony between TC and TRN is regulated by the burst firing of TRN neurons (10). However, there have been no direct experimental tests of the hypothesis that TRN bursts are indeed essential for SWDs in absence seizures. Here we have tested the role of TRN bursts in absence epilepsy and have surprisingly found that mice with complete genetic deletion of TRN burst activity exhibited enhanced SWDs with higher susceptibility to γ-butyrolactone (GBL), a widely used seizure-inducing drug. Apparently that enhanced TRN tonic firing is observed in the absence of bursts is sufficient to support SWDs.     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)_2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT_2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT_2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT_2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT_2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions.The prefrontal cortex (PFC) is a brain region critical for many high-level cognitive processes, such as executive functions, attention, and working and contextual memories (1). Pyramidal neurons located in layer V of the PFC integrate excitatory glutamatergic inputs originating from both cortical and subcortical areas. The latter include the mediodorsal thalamus (MD) nuclei, which project densely to the medial PFC (mPFC) and are part of the neuronal network underlying executive control and working memory (2–4). Disruption of this network has been involved in cognitive symptoms of psychiatric disorders, such as schizophrenia (3, 5). These symptoms severely compromise the quality of life of patients and remain poorly controlled by currently available antipsychotics (3, 6).The PFC is densely innervated by serotonin (5-hydroxytryptamine, 5-HT) neurons originating from the dorsal and median raphe nuclei and numerous lines of evidence indicate a critical role of 5-HT in the control of emotional and cognitive functions depending on PFC activity (7, 8). The modulatory action of 5-HT reflects its complex pattern of effects on cortical network activity, depending on the 5-HT receptor subtypes involved, and on receptor localization in pyramidal neurons, GABAergic interneurons or nerve terminals of afferent neurons.Among the 14 5-HT receptor subtypes, the 5-HT_2A receptor is a Gq protein-coupled receptor (9, 10) particularly enriched in the mPFC, with a predominant expression in apical dendrites of layer V pyramidal neurons (11–14). Moreover, a low proportion of 5-HT_2A receptors was detected presynaptically on thalamocortical fibers (12, 15–17).Activation of 5-HT_2A receptors exerts complex effects upon the activity of the PFC network (18). The most prominent one is an increase in pyramidal neuron excitability, which likely results from the inhibition of slow calcium-activated after hyperpolarization current (19). 5-HT_2A receptor stimulation also increases the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons (19–22). The prevailing view is that postsynaptic 5-HT_2A receptors expressed on pyramidal neurons located in layer V are key modulators of glutamatergic PFC network activity (14, 21–24). However, the role of presynaptic 5-HT_2A receptors located on thalamic afferents in the modulation of glutamatergic transmission at thalamocortical synapses remains unexplored.Here, we addressed this issue by combining electrophysiological recordings in acute PFC slices with viral infections to specifically rescue the expression of 5-HT_2A receptors at the presynaptic site (MD) or postsynaptic site (PFC) in 5-HT_2A receptor-deficient (5-HT_2A^−/−) mice (25). We focused our study on NMDA transmission in line with previous findings indicating that many symptoms of schizophrenia might arise from modifications in PFC connectivity involving glutamatergic transmission at NMDA receptors (26, 27). To our knowledge, we provide the first direct evidence that stimulation of presynaptic 5-HT_2A receptors at thalamocortical synapses gates the induction of spike timing-dependent long-term depression (t-LTD) by facilitating the activation of presynaptic NMDA receptors at these synapses. In line with the role of t-LTD in associative learning (28), these studies were extended by behavioral experiments to explore the role of presynaptic 5-HT_2A receptors at thalamocortical synapses in several paradigms of episodic-like memory.     

Biphasic regulation of InsP3 receptor gating by dual Ca2+ release channel BH3-like domains mediates Bcl-xL control of cell viability

Antiapoptotic Bcl-2 family members interact with inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channels in the endoplasmic reticulum to modulate Ca²⁺ signals that affect cell viability. However, the molecular details and consequences of their interactions are unclear. Here, we found that Bcl-x_L activates single InsP₃R channels with a biphasic concentration dependence. The Bcl-x_L Bcl-2 homology 3 (BH3) domain-binding pocket mediates both high-affinity channel activation and low-affinity inhibition. Bcl-x_L activates channel gating by binding to two BH3 domain-like helices in the channel carboxyl terminus, whereas inhibition requires binding to one of them and to a previously identified Bcl-2 interaction site in the channel-coupling domain. Disruption of these interactions diminishes cell viability and sensitizes cells to apoptotic stimuli. Our results identify BH3-like domains in an ion channel and they provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca²⁺ signaling and cell viability.The inositol trisphosphate receptors (InsP₃R) are a family of intracellular cation channels that release Ca²⁺ from the endoplasmic reticulum (ER) in response to a variety of extracellular stimuli (1). Three InsP₃R isoforms are ubiquitously expressed and regulate diverse cell processes, including cell viability (1). Activation of the channels by InsP₃ elicits changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that provide versatile signals to regulate molecular processes with high spatial and temporal fidelity (1). Regions of close proximity to mitochondria enable localized Ca²⁺ release events to be transduced to mitochondria (2, 3). Ca²⁺ released from the ER during cell stimulation modulates activities of effector molecules and is taken up by mitochondria to stimulate oxidative phosphorylation and enhance ATP production (4–6) to match energetic supply with enhanced demand. In addition, cells in vivo are constantly exposed to low levels of circulating hormones, transmitters, and growth factors that bind to plasma membrane receptors to provide a background level of cytoplasmic InsP₃ (7) that generates low-level stochastic InsP₃R-mediated localized or propagating [Ca²⁺]_i signals (8–10). Such signals also play an important role in maintenance of cellular bioenergetics (8). Nevertheless, under conditions of cell stress the close proximity of mitochondria to Ca²⁺ release sites may result in mitochondrial Ca²⁺ overload and initiate Ca²⁺-dependent forms of cell death, including necrosis and apoptosis (11–13). It has been suggested that high levels of ER Ca²⁺ (14–16) and enhanced activity of the InsP₃R (17–19) promote cell death by providing a higher quantity of released Ca²⁺ to mitochondria (3, 20, 21).Protein interactions modulate the magnitude and quality of InsP₃R-mediated [Ca²⁺]_i signals that regulate apoptosis and cell viability. Notable in this regard is the Bcl-2 protein family. Proapoptotic Bcl-2–related proteins Bax and Bak initiate cytochrome C release from mitochondria in response to diverse apoptotic stimuli, whereas antiapoptotic Bcl-2–related proteins, including Bcl-2 and Bcl-x_L, antagonize Bax/Bak by forming heterodimers that prevent their oligomerization and apoptosis initiation (22, 23). Heterodimerization is mediated by interactions of proapoptotic Bcl-2 homology 3 (BH3) domains with a hydrophobic groove on the surface of antiapoptotic Bcl-2 proteins (23) that is a therapeutic target in diseases, including cancer (22). Whereas a central feature of molecular models of apoptosis is the control of outer mitochondrial membrane permeability by Bcl-2–related proteins, a substantial body of evidence has demonstrated that these proteins localize to the ER (24, 25), bind to InsP₃Rs (26–32) and, by modulating InsP₃R-mediated Ca²⁺ release, regulate ER-mediated cell death and survival (15, 27, 32–34). Nevertheless, a unified understanding of the detailed molecular mechanisms by which Bcl-2 family proteins interact with and regulate InsP₃R channel activity is lacking. The Bcl-2 family member homolog NrZ interacts with the amino-terminal InsP₃-binding region via its helix 1 BH4 domain and inhibits Ca²⁺ release (28). Bcl-2 also interacts with the InsP₃R (26) via its BH4 domain (35), but in contrast it associates with a region in the central coupling domain (35). Whereas this interaction also inhibits Ca²⁺ release (26), Bok interacts with the channel 500 residues C-terminal to the Bcl-2 binding sequence via its BH4 domain but does not affect Ca²⁺ release (29). Conversely, the Bcl-x_L BH4 domain may lack this interaction (36). Inhibition of the Bcl-2 BH4 domain interaction with the channel enhanced InsP₃R-mediated Ca²⁺ signals and apoptosis sensitivity in white blood cells (18, 35, 37). However, it is unclear if Bcl-2 inhibits Ca²⁺ signaling directly by binding to the channel or if it acts indirectly, as a hub in a protein complex that influences channel phosphorylation (38). Conversely, we demonstrated that Bcl-x_L, Bcl-2, and Mcl-1 bind to the carboxyl (C)-terminus of all three InsP₃R isoforms, and showed that these interactions activated single InsP₃R channels and promoted InsP₃R-mediated Ca²⁺ release and apoptosis resistance (27, 31, 32). Furthermore, Bcl-x_L mediates an interaction of oncogenic K-RAS with the InsP₃R C terminus that regulates its biochemical and functional interaction and cell survival (39). However, the molecular details of the interactions of antiapoptotic protein with the InsP₃R C terminus are unknown. Furthermore, the relationship between Bcl-2 family protein binding in the coupling domain and C terminus is unclear. Thus, the mechanisms whereby Bcl-2 and Bcl-x_L affect InsP₃R activity and the effects of this modulation on cell viability remain to be determined.Here, we used single-channel electrophysiology of native ER membranes to explore the detailed mechanisms of the effects of Bcl-x_L on the InsP₃R, and the role of this interaction on cell viability. Surprisingly, our results reveal that whereas Bcl-x_L activates the channel at low concentrations, it inhibits it at higher concentrations, resulting in a biphasic response of channel activation on [Bcl-x_L]. Remarkably, the Bcl-x_L BH3 domain-binding pocket is required for both effects. Low [Bcl-x_L] activates the channel by simultaneous binding to two BH3 domain-like helices in the channel C terminus, whereas channel inhibition at high [Bcl-x_L] requires binding to only one of them and to a site previously identified as the Bcl-2 binding site in the channel-coupling domain. Disruption of these interactions diminishes cell viability. Our results provide a unifying model of the effects of antiapoptotic Bcl-2 proteins on the InsP₃R that play critical roles in Ca²⁺ signaling and cell viability.     

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons

Short-term synaptic plasticity is induced by calcium (Ca²⁺) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated Ca_V2.1 Ca²⁺ channels by Ca²⁺ sensor proteins induces facilitation of Ca²⁺ currents and synaptic facilitation in cultured neurons expressing exogenous Ca_V2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca²⁺ sensor binding site. This mutation does not alter voltage dependence or kinetics of Ca_V2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼50%. In the presence of EGTA-AM to prevent global increases in free Ca²⁺, the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca²⁺ is dependent upon regulation of Ca_V2.1 channels by Ca²⁺ sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10–20 Hz. Evidently, regulation of Ca_V2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.Modification of synaptic strength in central synapses is highly dependent upon presynaptic activity. The frequency and pattern of presynaptic action potentials regulates the postsynaptic response through diverse forms of short- and long-term plasticity that are specific to individual synapses and depend upon accumulation of intracellular Ca²⁺ (1–4). Presynaptic plasticity regulates neurotransmission by varying the amount of neurotransmitter released by each presynaptic action potential (1–5). P/Q-type Ca²⁺ currents conducted by voltage-gated Ca_V2.1 Ca²⁺ channels initiate neurotransmitter release at fast excitatory glutamatergic synapses in the brain (6–9) and regulate short-term presynaptic plasticity (3, 10). These channels exhibit Ca²⁺-dependent facilitation and inactivation that is mediated by the Ca²⁺ sensor (CaS) protein calmodulin (CaM) bound to a bipartite site in their C-terminal domain composed of an IQ-like motif (IM) and a CaM binding domain (CBD) (11–14). Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents correlate with facilitation and rapid depression of synaptic transmission at the Calyx of Held (15–18). Elimination of Ca_V2.1 channels by gene deletion prevents facilitation of synaptic transmission at the Calyx of Held (19, 20). Cultured sympathetic ganglion neurons with presynaptic expression of exogenous Ca_V2.1 channels harboring mutations in their CaS regulatory site have reduced facilitation and slowed depression of postsynaptic responses because of reduced Ca²⁺-dependent facilitation and Ca²⁺-dependent inactivation of Ca_V2.1 currents (21). The CaS proteins Ca²⁺-binding protein 1 (CaBP-1), visinin-like protein-2 (VILIP-2), and neuronal Ca²⁺ sensor-1 (NCS-1) induce different degrees of Ca²⁺-dependent facilitation and inactivation of channel activity (22–26). Expression of these different CaS proteins with Ca_V2.1 channels in cultured sympathetic ganglion neurons results in corresponding bidirectional changes in facilitation and depression of the postsynaptic response (25, 26). Therefore, binding of CaS proteins to Ca_V2.1 channels at specific synapses can change the balance of CaS-dependent facilitation and inactivation of Ca_V2.1 channels, and determine the outcome of synaptic plasticity (27). Currently, it is not known whether such molecular regulation of Ca_V2.1 by CaS proteins induces or modulates synaptic plasticity in native hippocampal synapses.To understand the functional role of regulation of Ca_V2.1 channels by CaS proteins in synaptic plasticity in vivo, we generated knock-in mice with paired alanine substitutions for the isoleucine and methionine residues in the IM motif (IM-AA) in their C-terminal domain. Here we investigated the effects of mutating this CaS regulatory site on hippocampal neurotransmission and synaptic plasticity. This mutation had no effect on basal Ca²⁺ channel function or on basal synaptic transmission. However, we found reduced short-term facilitation in response to paired stimuli in autaptic synapses in hippocampal cultures and in Schaffer collateral (SC)-CA1 synapses in acutely prepared hippocampal slices. Moreover, synaptic facilitation in mutant SC-CA1 synapses developed and decayed more slowly during trains of stimuli. These results identify a critical role for modulation of Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity, which is likely to have important consequences for encoding and transmitting information in the hippocampus.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Proteins containing C2 domains are the sensors for Ca²⁺ and PI(4,5)P₂ in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca²⁺ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P₂ and IP₃ at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3–β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P₂/PI(3,4,5)P₃, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P₂. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca²⁺-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1–4). Many of these C2 domains have been demonstrated to function in a Ca²⁺-dependent membrane-binding manner and hence act as cellular Ca²⁺ sensors. Calcium ions bind in a cup-shaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca²⁺ ions are incomplete (5–7). This incomplete coordination sphere can be occupied by neutral and anionic (7–9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P₂ simultaneously (10). This revealed an additional lipid-binding site located in the polybasic region formed by β3–β4 strands that preferentially binds to PI(4,5)P₂ (11–15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10, 16–19). Given the importance of PI(4,5)P₂ for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20–23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca²⁺ and PI(4,5)P₂ combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16, 20, 22, 24–28). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5, 26, 27, 29, 30). However, the impossibility of obtaining crystal structures of these domains in complex with Ca²⁺ and phosphoinositides has hindered the understanding of the molecular mechanism driving the PI(4,5)P₂–C2 domain interaction. Here, we sought to unravel the molecular mechanism of Ca²⁺ and PI(4,5)P₂ binding to the C2A domain of rabphilin 3A by X-ray crystallography. A combination of site-directed mutagenesis together with isothermal titration calorimetry (ITC), fluorescence resonance of energy transfer (FRET), and aggregation experiments has enabled us to propose a molecular mechanism of Ca²⁺/PI(4,5)P₂-dependent membrane interaction through two different motifs that could bend the membrane and accelerate the vesicle fusion process. A comparative analysis revealed the structural basis for the different phosphoinositide affinities of C2A and -B domains. Furthermore, the C2A domain of synaptotagmin 1 lacks one of the key residues responsible for the PI(4,5)P₂ interaction, confirming it is a non-PI(4,5)P₂ responder.     

STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺ sensor, is unclear with respect to its cellular localization, its Ca²⁺-dependent mobilization, and its action on Ca²⁺ signaling. Confocal microscopy was used to measure Ca²⁺ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca²⁺ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca²⁺ depletion. Additionally, we found no store-operated Ca²⁺ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca²⁺ content and increased SR Ca²⁺ leak. These changes in Ca²⁺ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca²⁺ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca²⁺ leak and that these actions are independent of store-operated Ca²⁺ entry, a process that is absent in normal heart cells.Store-operated Ca²⁺ entry (SOCE) is a cellular mechanism to ensure that sufficient levels of Ca²⁺ are present in the intracellular Ca²⁺ stores to enable robust signaling (1). SOCE depends on the presence and interaction of two proteins, STIM1 (stromal interaction molecule 1) and Orai1 (a low conductance plasma/sarcolemmal Ca²⁺ channel), or their equivalents (2–5). STIM1 is an endo/sarcoplasmic reticulum (ER/SR) Ca²⁺-sensitive protein that interacts with Orai1 to activate the channel function of Orai1, a Ca²⁺ selective channel, and thus permit Ca²⁺ entry. SOCE is clearly present in nonexcitable cells such as T lymphocytes and some excitable cells including skeletal muscle cells (4, 6–13). STIM1 is a membrane-spanning ER/SR protein with a single transmembrane domain and a luminal Ca²⁺ ([Ca²⁺]_ER/SR)-sensing domain. When luminal Ca²⁺ is low (i.e., [Ca²⁺]_ER/SR drops to less than 300 µM), then STIM1 self-aggregates and associates with Orai1 to activate it, producing a SOCE current (I_SOCE) (2, 14–16) and Ca²⁺ entry (with a reversal potential E_SOCE ∼ +50 mV or more) (17, 18). Then, as [Ca²⁺]_ER/SR increases in response to the Ca²⁺ influx, the process reverses.In adult skeletal muscle cells, Ca²⁺ influx is normally low, and it has been suggested that SOCE is needed for maintaining an appropriate level of [Ca²⁺]_ER/SR and correct Ca²⁺ signaling (6, 7, 9, 19). In skeletal muscle, it has been hypothesized that STIM1 is prelocalized in the SR terminal cisternae (6, 20) and hence can more rapidly respond to changes in [Ca²⁺]_ER/SR. The putative importance of SOCE in skeletal muscle was further supported by the observation that the skeletal muscle dysfunction is significant in STIM1-null mice where 91% (30/33) of the animals died in the perinatal period from a skeletal myopathy (6). Furthermore, in humans, STIM1 mutations were identified as a genetic cause of tubular aggregate myopathy (21).Despite the clarity of the SOCE paradigm, the canonical SOCE activation process described above does not apply to all conditions in which STIM1 and Orai1 interact. For example, in T lymphocytes, STIM1 clustering is necessary and sufficient to activate SOCE, regardless of whether [Ca²⁺]_ER/SR is low (4). When present, the STIM1 EF hand mutation causes STIM1 oligomerization and constitutive Ca²⁺ influx across the plasma membrane into cells with full Ca²⁺ stores (4). Although this is consistent with the use of STIM1 clusters and puncta to measure the activation of Orai1 (15, 16, 22, 23), it does not necessarily reflect the state of [Ca²⁺]_ER/SR. Furthermore, several small-molecule bioactive reagents, such as 2-APB and FCCP, neither of which causes [Ca²⁺]_ER/SR depletion, induce STIM1 clustering (24). Thus, STIM1 may have actions that are more complicated than simple [Ca²⁺]_ER/SR sensing and Orai1 signaling.Cardiomyocytes have been reported to have SOCE (8, 13, 25, 26) but are very different from many of the cells noted above that exhibit significant [Ca²⁺]_ER/SR depletion-sensitive Ca²⁺ entry through the Ca²⁺-selective Orai1. Cardiac ventricular myocytes are different from the other cells in that they have large, regular, and dynamic changes in [Ca²⁺]_i and robust influx and extrusion pathways across the sarcolemmal membrane. For example, it is not unusual for investigators to measure a 10–20 nA calcium current (I_Ca,L) in single cardiac ventricular myocytes that is readily extruded by the sarcolemmal Na⁺/Ca²⁺ exchanger. Because of these large fluxes, adult ventricular myocytes have no “need” for SOCE and the same logic applies to neonatal cardiomyocytes. Nevertheless, reports of SOCE in neonatal cardiac myocytes are clear (10, 12, 13). Against this background, we have attempted to determine if STIM1 is present in adult cardiomyocytes and, if so, where the protein is located, how it is mobilized, and how it may interact with other Ca²⁺ signal proteins. In the work presented here, we show that STIM1 is present but that its function in heart is distinct from the canonical SOCE behavior and does not contribute to Ca²⁺ influx through I_SOCE. Instead we show that STIM1 binds phospholamban (PLN), an endogenous SERCA2a inhibitor in the heart (27), and by doing so reduces the PLN-dependent inhibition of SERCA2a and thereby indirectly activates SERCA2a.     

Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors

The inositol 1,4,5-trisphosphate receptor (IP₃R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP₃Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP₃ at a distance. Defective allostery in IP₃R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP₃R type 1 (IP₃R1) and negatively regulates IP₃R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP₃R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.Ligand-gated ion channels function by allostery that is the regulation at a distance; the allosteric coupling of ligand binding with channel gating requires reversible changes in subunit configurations and conformations (1). Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are ligand-gated ion channels that release calcium ions (Ca²⁺) from the endoplasmic reticulum (ER) (2, 3). IP₃Rs are allosteric proteins comprising four subunits that assemble a calcium channel with fourfold symmetry about an axis perpendicular to the ER membrane. The subunits of three IP₃R isoforms (IP₃R1, IP₃R2, and IP₃R3) are structurally divided into three domains: the IP₃-binding domain (IBD), the regulatory domain, and the channel domain (3–6). Fitting of the IBD X-ray structures (7, 8) to a cryo-EM map (9) indicates that the IBD activates a remote Ca²⁺ channel by allostery (8); however, the current X-ray structure only spans 5% of each tetramer, such that the mechanism underlying allosteric coupling of the IBD to channel gating remains unknown.The IP₃R in the ER mediates intracellular calcium signaling that impinges on homeostatic control in various subsequent intracellular processes. Deletion of the genes encoding the type 1 IP₃R (IP₃R1) leads to perturbations in long-term potentiation/depression (3, 10, 11) and spinogenesis (12), and the human genetic disease spinocerebellar ataxia 15 is caused by haploinsufficiency of the IP₃R1 gene (13–15). Dysregulation of IP₃R1 is also implicated in neurodegenerative diseases including Huntington disease (HD) (16–18) and Alzheimer’s disease (AD) (19–22). IP₃Rs also control fundamental cellular processes—for example, mitochondrial energy production (23, 24), autophagy regulation (24–27), ER stress (28), hepatic gluconeogenesis (29), pancreatic exocytosis (30), and macrophage inflammasomes (31). On the other hand, excessive IP₃R function promotes cell death processes including apoptosis by activating mitochondrial or calpain pathways (2, 17). Considering these versatile roles of IP₃Rs, appropriate IP₃R structure and function are essential for living systems, and aberrant regulation of IP₃R closely relates to various diseases.Several factors such as cytosolic molecules, interacting proteins, and posttranslational modifications control the IP₃-induced Ca²⁺ release (IICR) through allosteric sites in IP₃Rs. Cytosolic Ca²⁺ concentrations strictly control IICR in a biphasic manner with activation at low concentrations and inhibition at higher concentrations. The critical Ca²⁺ sensor for activation is conserved among the three isoforms of IP₃ and ryanodine receptors, and this sensor is located in the regulatory domain outside the IBD and the channel domain (32). A putative ATP regulatory region is deleted in opisthotonos mice, and IICR is also regulated by this mutation in the regulatory domain (33). Various interacting proteins, such as cytochrome c, Bcl-2-family proteins, ataxin-3, huntingtin (Htt) protein, Htt-associated protein 1A (HAP1A), and G-protein–coupled receptor kinase-interacting protein 1 (GIT1), target allosteric sites in the carboxyl-terminal tail (3–5). The regulatory domain and the carboxyl-terminal tail also undergo phosphorylation by the protein kinases A/G and B/Akt and contain the apoptotic cleavage sites for the protease caspase-3 (4, 5). These factors allosterically regulate IP₃R structure and function to control cellular fates; therefore, understanding the allosteric coupling of the IBD to channel gating will elucidate the regulatory mechanism of these factors.Transglutaminase (TG) catalyses protein cross-linking between a glutamine (Gln) residue and a lysine (Lys) residue via an N^ε-(γ-glutamyl)lysine isopeptide bond (34, 35). TG type 2 (TG2) is a Ca²⁺-dependent enzyme with widespread distribution and is highly inducible by various stimulations such as oxidative stress, cytokines, growth factors, and retinoic acid (RA) (34, 35). TG2 is considered a significant disease-modifying factor in neurodegenerative diseases including HD, AD, and Parkinson’s diseases (PD) (34, 36–45) because TG2 might enzymatically stabilize aberrant aggregates of proteins implicated in these diseases—that is, mutant Htt, β-amyloid, and α-synuclein; however, the causal role of TG2 in Ca²⁺ signaling in brain pathogenesis has been unclear. Ablation of TG2 in HD mouse models is associated with increased lifespan and improved motor function (46, 47). However, TG2 knockout mice do not show impaired Htt aggregation, suggesting that TG2 may play a causal role in these disorders rather than TG2-dependent cross-links in aberrant protein aggregates (47, 48).In this study, we discovered a new mode of chronic and irreversible allosteric regulation in IP₃R1 in which covalent modification of the receptor at Gln2746 is catalyzed by TG2. We demonstrate that up-regulation of TG2 modifies IP₃R1 structure and function in HD models and propose an etiologic role of this modification in the reduction of neuronal signaling and subsequent processes during the prodromal state of the neurodegenerative disease.     

Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca²⁺ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca²⁺ channels in pollen tube tips are core components that regulate Ca²⁺ gradients by mediating and regulating external Ca²⁺ influx. Therefore, Ca²⁺ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca²⁺ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca²⁺ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca²⁺ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca²⁺ channel for pollen tube guidance in Arabidopsis.Pollen tubes deliver paired sperm cells into ovules for double fertilization, and signaling communication between pollen tubes and female reproductive tissues is required to ensure the delivery of sperm cells into the ovules (1). Pollen tube guidance is governed by both female sporophytic and gametophytic tissues (2, 3) and can be separated into two categories: preovular guidance and ovular guidance (1). For preovular guidance, diverse signaling molecules from female sporophytic tissues have been identified, including the transmitting tissue-specific (TTS) glycoprotein in tobacco (4), γ-amino butyric acid (GABA) in Arabidopsis (5), and chemocyanin and the lipid transfer protein SCA in Lilium longiflorum (6, 7). For ovular pollen tube guidance, female gametophytes secrete small peptides as attractants, including LUREs in Torenia fournieri (8) and Arabidopsis (9) and ZmEA1 in maize (10, 11). Synergid cells, central cells, egg cells, and egg apparatus are all involved in pollen tube guidance, probably by secreting different attractants (9–15). Additionally, nitric oxide (NO) and phytosulfokine peptides have also been implicated in both preovular and ovular pollen tube guidance (16–18). Thus, pollen tubes could be guided by diverse attractants in a single plant species.Ca²⁺ gradients at pollen tube tips are essential for both tip growth and pollen tube guidance (19–27). Spatial modification of the Ca²⁺ gradients leads to the reorientation of pollen tube growth in vitro (28, 29). The Ca²⁺ gradients were significantly increased in pollen tubes attracted to the micropyles by synergid cells in vivo, compared with those not attracted by ovules (30). Therefore, the Ca²⁺ gradients in pollen tube tips are essential for pollen tube guidance. The Ca²⁺ gradients result from external Ca²⁺ influx, which is mainly mediated by plasma membrane Ca²⁺ channels in pollen tube tips. Thus, the Ca²⁺ channels are the key components for regulating the Ca²⁺ gradients and are consequently essential for pollen tube guidance. Using electrophysiological techniques, inward Ca²⁺ currents were observed in both pollen grain and pollen tube protoplasts (31–36), supporting the presence of plasma membrane Ca²⁺ channels in pollen tube tips. Recently, a number of candidate Ca²⁺ channels were identified in pollen tubes, including six cyclic nucleotide-gated channels (CNGCs) and two glutamate receptor-like channels (GLRs) in Arabidopsis (37–40). Three of these eight channels, namely CNGC18, GLR1.2, and GLR3.7, were characterized as Ca²⁺-permeable channels (40, 41) whereas the ion selectivity of the other five CNGCs has not been characterized. We hypothesized that the Ca²⁺ channel essential for pollen tube guidance could be among these eight channels.In this research, we first characterized the remaining five CNGCs as Ca²⁺ channels. We further found that CNGC18, out of the eight Ca²⁺ channels, was the only one essential for pollen tube guidance in Arabidopsis and that its transmembrane domains were indispensable for pollen tube guidance.     

UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling

At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca²⁺ response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.The surface epithelium (epidermis) of skin provides barrier protection against dehydration and the potentially harmful external environment (1). Accordingly, skin is the site of first interaction between ambient environment and immunologically competent organismal structures, and also the site for sentient responses (2). Sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) are endowed with sensory transduction capacity for heat, cold, mechanical cues, itch, and pain, and their axons directly interface with skin epithelium (2–4).Against a background of suggestive findings (2, 5–7), we wondered whether the epidermis as a “forefront” of sensory signaling may function in sensitizing pain transduction in response to naturally occurring irritating cues. To elucidate mechanisms, we used a mouse sunburn model and induced a state of lowered sensory thresholds associated with tissue injury caused by UV radiation (8–10). UV-sunburn-evoked lowering of sensory thresholds shares major hallmarks of pathological pain, a valuable feature of this model. Skin tissue injury caused by UVB has been elucidated to be mediated by cytokines and chemokines, known from immunological responses, such as IL-1β and IL-6, which are also known to cause and facilitate pain (11–19). Another more recent study identified a proinflammatory chemokine, CXCL5, as proalgesic in response to UVB overexposure of rat and human skin (20). An exciting new arena pertaining to molecular mechanisms of the skin’s response to noxious UV was recently opened by an elegant study that reported the role of UVB-mediated damage to noncoding RNA molecules in the skin (21). Unraveling a molecular mechanism, the Toll-like receptor 3 gene was found critical in signaling the proinflammatory actions of the UVB-damaged noncoding RNA molecules. However, this study focused on molecular mechanisms of acute inflammation in the skin.We intended to identify pain mechanisms that mediate the pain associated with UVB-mediated tissue injury. Pain in response to external environmental cues has been understood better because of scientific progress in the field of transient receptor potential (TRP) ion channels that have been found responsive to such cues, and which were found expressed in DRG and TG peripheral sensory neurons, which are the cells believed to be the primary transducers. Indeed, TRPV1, one of the founding members of the TRPV channel subfamily, has been identified as relevant for pain, including pathological pain, response to thermal cues, and most recently for itch (22–31). However, TRPA1 (transient receptor potential ion channel, ankyrin subfamily, family member #1) and TRPM8 seem to be involved in transduction of pain-inducing stimuli as well (32–36).Also a family member of the TRPV subfamily, TRPV4 is a multimodally activated, nonselective cation channel that is involved in physiological pain evoked by osmotic and mechanical, but not thermal, cues (37–40). For pathological pain, it is relevant for inflammation- and nerve-damage-induced pain sensitization (41–43). Of note, Trpv4^−/− mice exhibit impaired skin-barrier function (44, 45). That said, TRPV4 is expressed in a number of different cell types, including robust expression in epidermal keratinocytes and also is detectable in skin-innervating sensory neurons. This “dual-location expression” of TRPV4 leaves the cellular mechanisms involved in the channel’s function and the functional contribution of environment-exposed keratinocytes vs. skin-innervating sensory neurons unclear.Against this background of dual-location TRPV4 expression and the role of TRPV4 in inflammatory and neuropathic pain, we now address whether epidermally derived TRPV4 is pathophysiologically relevant in sunburn pain and tissue damage. Using Trpv4 gene-targeted mice, selectively inducing targeting in postnatal keratinocytes, and topically applying selective TRPV4 inhibitors, we demonstrate that epidermal TRPV4 plays a prominent, hitherto unrecognized role in UVB-evoked skin tissue damage and pain of sunburn.     

In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor

cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca²⁺ biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O₂ can evoke a tonic increase in cGMP that requires an atypical O₂-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca²⁺ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca²⁺ stimulate competing negative feedback loops that shape cGMP dynamics. Ca²⁺-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O₂, apparently by keeping in check gating of cGMP channels and limiting activation of Ca²⁺-dependent negative feedback loops. Simultaneous imaging of Ca²⁺ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O₂-evoked cGMP and Ca²⁺ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling.The second messenger cyclic guanosine monophosphate (cGMP) regulates a range of physiological processes. In nervous systems, it can transduce sensory inputs (1) and modulate neuronal excitability and learning (2) and is implicated in control of mood and cognition (3). Precise regulation of cGMP levels ([cGMP]) is thought critical for these functions. This importance has prompted development of genetically encoded cGMP indicators, with the goal of visualizing cGMP dynamics with high temporal and spatial resolution (4, 5). Although these sensors have been used to image pharmacologically evoked changes in cGMP in cultured cells or tissue slices (6–10), endogenous cGMP dynamics have not been visualized and functionally dissected in vivo in any nervous system (4, 5).Local [cGMP] reflects the net activity of guanylate cyclases (GCs) that synthesize cGMP (11) and phosphodiesterases (PDEs) that degrade it (12, 13). Mammals have several families of GCs (14, 15) and eight families of cGMP PDEs (16), each with distinct regulatory properties. Different PDE types are often coexpressed, but little is known about how they work together. cGMP signaling alters cell physiology by controlling cGMP-dependent protein kinases (PKG) (17, 18), cGMP-gated channels (CNGC) (19), and cGMP-regulated PDEs (12). These cGMP effectors can also feed back to control cGMP dynamics.cGMP is a major second messenger in Caenorhabditis elegans, implicated in the function of a third of its sensory neurons, including thermosensory, olfactory, gustatory, and O₂-sensing neurons (20). Genetic and behavioral studies suggest that cGMP mediates sensory transduction in many of these neurons (21–25). Despite this pervasiveness, cGMP has not been visualized in any C. elegans cell: genetic inferences about its roles in signal transduction are untested, and we have no mechanistic insights into cGMP signaling dynamics and feedback control. Consistent with the prominence of cGMP signaling in the nematode, the C. elegans genome encodes 34 GCs (26), six PDE genes, at least one PKG (24, 27, 28), and six CNGC subunits (19, 21, 22, 29–31).Here, we use cGMP and Ca²⁺ sensors to visualize and dissect cGMP signaling dynamics in a C. elegans O₂ sensor. We image single and double mutants defective in a soluble guanylate cyclase (sGC), CNGC subunits, PDE-1, PDE-2, and PKG. Our results reveal a signaling network of interwoven checks and balances. Counterintuitively, cGMP activation of PDE-2 promotes cGMP accumulation by controlling gating of CNGC and limiting Ca²⁺-mediated negative feedback, including activation of PDE-1. We show that cGMP signal transduction is highly reliable when the same individual is stimulated repeatedly but, surprisingly, is highly variable across genetically identical, similarly reared animals. Finally, simultaneous imaging of O₂-evoked cGMP and Ca²⁺ responses suggests that cGMP dynamics can differ in distinct subcellular compartments of a C. elegans neuron, consistent with the existence of cGMP nanodomains.     

Long-term potentiation of glycinergic synapses triggered by interleukin 1β

Long-term potentiation (LTP) is a persistent increase in synaptic strength required for many behavioral adaptations, including learning and memory, visual and somatosensory system functional development, and drug addiction. Recent work has suggested a role for LTP-like phenomena in the processing of nociceptive information in the dorsal horn and in the generation of central sensitization during chronic pain states. Whereas LTP of glutamatergic and GABAergic synapses has been characterized throughout the central nervous system, to our knowledge there have been no reports of LTP at mammalian glycinergic synapses. Glycine receptors (GlyRs) are structurally related to GABA_A receptors and have a similar inhibitory role. Here we report that in the superficial dorsal horn of the spinal cord, glycinergic synapses on inhibitory GABAergic neurons exhibit LTP, occurring rapidly after exposure to the inflammatory cytokine interleukin-1 beta. This form of LTP (GlyR LTP) results from an increase in the number and/or change in biophysical properties of postsynaptic glycine receptors. Notably, formalin-induced peripheral inflammation in vivo potentiates glycinergic synapses on dorsal horn neurons, suggesting that GlyR LTP is triggered during inflammatory peripheral injury. Our results define a previously unidentified mechanism that could disinhibit neurons transmitting nociceptive information and may represent a useful therapeutic target for the treatment of pain.Glycine mediates fast synaptic inhibition throughout the spinal cord, brainstem, and midbrain, controlling normal motor behavior and rhythm generation, somatosensory processing, auditory and retinal signaling, and coordination of reflex responses (1). Strychnine-sensitive glycine receptors (GlyRs) are pentameric ligand-gated chloride channels of the Cys-loop receptor family that together with GABA_A receptors (GABA_ARs) dynamically interact with the synaptic scaffold protein gephyrin to form inhibitory synapses (1, 2). In the dorsal horn of the spinal cord, glycinergic synapses are essential for nociceptive and tactile sensory processing both during adaptive and pathological pain states (3–7). However, compared with glutamatergic and GABAergic synapses, little is known about the regulation of their synaptic strength. Several studies have examined glycine receptor trafficking in cultured neurons and in heterologous expression systems (8, 9). Intracellular Ca²⁺ appears important in the stabilization of GlyRs at synapses in culture (10), and elevation of intracellular Ca²⁺ can also potently increase glycine receptor single channel openings in cultured cells and in heterologous systems (11). However, the modulation of glycinergic synaptic strength in native tissue remains relatively unexplored.Following peripheral injury or inflammation, changes in tactile perception develop, including hyperalgesia (exaggerated pain upon noxious stimulation), allodynia (pain in response to innocuous stimuli), and secondary hyperalgesia (pain spreading beyond the confines of the injured region). Inhibitory interneurons of the spinal dorsal horn have been proposed to gate the flow of innocuous and nociceptive sensory information from the periphery to higher brain centers (12), and supportive evidence for this idea is growing (13–17). Loss of GABAergic/glycinergic inhibition contributes to enhanced transmission of nociceptive signals through the dorsal horn circuit during pain states, resulting in hyperalgesia and allodynia (3, 18–20). For example, polysynaptic A-fiber inputs onto neurokinin 1 receptor (NK1R)-expressing projection neurons become apparent only when GABA_AR and GlyRs are pharmacologically blocked, indicating that under conditions of disinhibition, nonnoxious mechanical stimuli can drive nociceptive-specific projection pathways and elicit allodynia (21). The majority of neurons tested in the dorsal horn receive glycinergic synapses, including lamina I projection neurons, both excitatory and inhibitory interneurons of lamina II (22, 23), and inhibitory glycinergic neurons (24). Given the diversity of afferent targets, it is likely that glycinergic synapses are differentially modulated in a cell type- and subregion-specific manner. For example, during chronic inflammation, prostaglandin E₂ selectively depresses glycinergic synaptic inputs onto nonglycinergic neurons (24). Similarly, peripheral nerve injury suppresses glycinergic inhibition of a specific excitatory interneuron class [protein kinase C (PKC)γ⁺ neurons receiving Aβ fiber inputs], allowing excitatory afferents carrying nonnociceptive tactile information to activate ascending projections of nociceptive pathways that are normally under strong inhibitory control (23).Both hyperalgesia and allodynia occur within minutes of peripheral inflammation, but the mechanisms underlying these rapid perceptual alterations are poorly understood. The proinflammatory cytokine, IL-1β, is a potent hyperalgesic agent (25–27), contributing both to peripheral and central sensitization after tissue damage (28–31). Following tissue trauma, nerve injury, or inflammation, IL-1β levels are up-regulated in the spinal cord itself (29, 32, 33), and delivery of IL-1β intrathecally increases the activity of superficial dorsal horn neurons that transmit pain signals to the brain (34, 35). Intrathecal delivery of an IL-1 receptor antagonist blocks allodynia in rodent models of inflammatory pain (36, 37). A recent study also found that IL-1β application rapidly potentiated primary afferent (glutamatergic) synapses in dorsal horn slices, through unidentified signaling molecules released from glial cells (38). Here we report that IL-1β rapidly elicits a postsynaptic form of long-term potentiation (LTP) at glycinergic synapses on lamina II inhibitory neurons (GlyR LTP), and that the same glycinergic synapses are potentiated after peripheral inflammation.     

From the Cover: Mitochondrial calcium overload is a key determinant in heart failure

Calcium (Ca²⁺) released from the sarcoplasmic reticulum (SR) is crucial for excitation–contraction (E–C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca²⁺ is essential for optimal function of these organelles. However, Ca²⁺ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca²⁺ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca²⁺ leak causes mitochondrial Ca²⁺ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca²⁺ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca²⁺ leak, we found that leaky RyR2 channels result in mitochondrial Ca²⁺ overload, dysmorphology, and malfunction. In contrast, cardiac-specific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca²⁺ overload and dysfunction in HF.Type 2 ryanodine receptor/Ca²⁺ release channel (RyR2) and type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) are the major intracellular Ca²⁺ release channels in the heart (1–3). RyR2 is essential for cardiac excitation–contraction (E–C) coupling (2), whereas the role of IP3R2 in cardiomyocytes is less well understood (3). E–C coupling requires energy in the form of ATP produced primarily by oxidative phosphorylation in mitochondria (4–8).Both increased and reduced mitochondrial Ca²⁺ levels have been implicated in mitochondrial dysfunction and increased reactive oxygen species (ROS) production in heart failure (HF) (6, 7, 9–17). Albeit Ca²⁺ is required for activation of key enzymes (i.e., pyruvate dehydrogenase phosphatase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the tricarboxylic acid (also known as Krebs) cycle (18, 19), excessive mitochondrial Ca²⁺ uptake has been associated with cellular dysfunction (14, 20). Furthermore, the exact source of mitochondrial Ca²⁺ has not been clearly established. Given the intimate anatomical and functional association between the sarcoplasmic reticulum (SR) and mitochondria (6, 21, 22), we hypothesized that SR Ca²⁺ release via RyR2 and/or IP3R2 channels in cardiomyocytes could lead to mitochondrial Ca²⁺ accumulation and dysfunction contributing to oxidative overload and energy depletion.     

Preassociated apocalmodulin mediates Ca2+-dependent sensitization of activation and inactivation of TMEM16A/16B Ca2+-gated Cl? channels

Ca²⁺-activated chloride currents carried via transmembrane proteins TMEM16A and TMEM16B regulate diverse processes including mucus secretion, neuronal excitability, smooth muscle contraction, olfactory signal transduction, and cell proliferation. Understanding how TMEM16A/16B are regulated by Ca²⁺ is critical for defining their (patho)/physiological roles and for rationally targeting them therapeutically. Here, using a bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP)—we discovered that Ca²⁺-free calmodulin (apoCaM) is preassociated with TMEM16A/16B channel complexes. The resident apoCaM mediates two distinct Ca²⁺-dependent effects on TMEM16A, as revealed by expression of dominant-negative CaM₁₂₃₄. These effects are Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI). CDI and CDSA are independently mediated by the N and C lobes of CaM, respectively. TMEM16A alternative splicing provides a mechanism for tuning apoCaM effects. Channels lacking splice segment b selectively lost CDI, and segment a is necessary for apoCaM preassociation with TMEM16A. The results reveal multidimensional regulation of TMEM16A/16B by preassociated apoCaM and introduce ChIMP as a versatile tool to probe the macromolecular complex and function of Ca²⁺-activated chloride channels.Calcium (Ca²⁺)-activated chloride (Cl^–) channels (CaCCs) broadly expressed in mammalian cells regulate diverse physiological functions including: epithelial mucus secretion (1, 2), neuronal excitability (3–5), smooth muscle contraction (6), olfactory transduction (7, 8), and cell proliferation (9, 10). Drugs targeting CaCCs are being pursued as therapies for hypertension, cystic fibrosis, asthma, and cancer (1, 9, 11).Three laboratories independently identified the transmembrane protein TMEM16A as the molecular component of a CaCC (12–14). TMEM16A belongs to a protein family with 10 members encoded by distinct genes (15–18). There is universal agreement that TMEM16A, and the closely related TMEM16B, are bona fide CaCCs (2, 12–14, 19). Consistent with this, TMEM16A knockout mice displayed defective CaCC activity in a variety of epithelia (20–22), and the olfactory CaCC current was completely abolished in TMEM16B knockout mice (23). Hydropathy analyses suggest TMEM16 proteins have a similar topology with cytosolic N and C termini and eight predicted transmembrane helices (2, 19). Human TMEM16A has four alternatively spliced segments (a–d), differential inclusion of which modify voltage and Ca²⁺ sensitivity of resultant channel splice variants (24).CaCCs are highly sensitive to intracellular [Ca²⁺], displaying graded increases in Cl⁻ current (I_Cl) amplitude as [Ca²⁺]_i is raised from resting levels (∼100 nM) to the 1- to 2-μM range. In some cases, high [Ca²⁺]_i (>10 μM) leads to decreased I_Cl amplitude (inactivation) (25–27). The Ca²⁺ sensor(s) for Ca²⁺-dependent activation and inactivation (CDA and CDI) of TMEM16A/16B is unknown. There are two possible nonexclusive mechanisms: (i) direct Ca²⁺ binding to the channel or (ii) Ca²⁺ binding through a separate Ca²⁺-sensing protein. The TMEM16A sequence does not reveal any canonical Ca²⁺-binding EF hand motifs (14, 16, 17). A sequence in the first intracellular loop of TMEM16A resembling the “Ca²⁺ bowl” in large conductance Ca²⁺-activated K⁺ (BK) channels was disqualified by mutagenesis as the Ca²⁺ sensor responsible for CDA of TMEM16A (28). A revised TMEM16A topological model suggests the originally predicted extracellular loop 4 is located intracellularly (29), and mutating E702 and E705 within this loop markedly alter Ca²⁺ sensitivity of TMEM16A (29, 30).Some reports have suggested involvement of calmodulin (CaM) in distinct aspects of Ca²⁺-dependent regulation of CaCCs. Tian et al. reported that inhibiting CaM with trifluoperazine or J-8 markedly suppressed CDA of TMEM16A(abc) in HEK293 cells, and mapped the CaM binding site to splice segment b (31). They concluded that CaM is essential for TMEM16A activation. However, this suggestion is contradicted by the robust CDA of TMEM16A(ac), a splice variant lacking the putative CaM binding site on splice segment b (2, 24). Recently, Ca²⁺–CaM was found to bind TMEM16A(ac) in a Ca²⁺-dependent manner and result in an increased permeability of the channel to HCO₃⁻ (32). Deleting the Ca²⁺–CaM binding site did not affect CDA of TMEM16A(ac). Ca²⁺–CaM regulation of TMEM16A HCO₃⁻ permeability conforms to a traditional signaling mode where Ca²⁺ binds to freely diffusing CaM to form a Ca²⁺–CaM complex that then interacts with a target protein.There are several examples of an alternative mode of CaM signaling in which Ca²⁺-free CaM (apoCaM) is preassociated with target proteins under resting [Ca²⁺]_i conditions and acts as a resident Ca²⁺ sensor to regulate function of the host protein in response to increased [Ca²⁺]_i (33). This mode of CaM signaling is used as the activating mechanism for small conductance K⁺ channels (34) and Ca²⁺-dependent regulation of high voltage-gated Ca²⁺ (Ca_V1 and Ca_V2) channels (35, 36). A distinguishing feature of this mode of CaM signaling is that it is impervious to pharmacological inhibitors of Ca²⁺–CaM, but can be eliminated in dominant negative fashion by a CaM mutant, CaM₁₂₃₄, in which all four EF hands have been mutated so they no longer bind Ca²⁺ (34, 36). Whether apocalmodulin preassociates with TMEM16A/TMEM16B channel complexes and participates in Ca²⁺-dependent regulation of these channels is controversial (30, 37, 38).Using a recently developed bioengineering approach—channel inactivation induced by membrane-tethering of an associated protein (ChIMP) (39)—we discovered that apoCaM is functionally preassociated with TMEM16A/16B channel complexes. Whereas the resident apoCaM is not necessary for CDA, it does mediate two distinct Ca²⁺-dependent processes in TMEM16A. First, it causes a leftward shift in the Ca²⁺-dependent activation curve at [Ca²⁺]_i ≤ 1 μM, an effect we term Ca²⁺-dependent “sensitization” of activation (CDSA). Second, it is the Ca²⁺ sensor for CDI observed at [Ca²⁺]_i > 10 μM. The two opposite effects are independently mediated by the two lobes of preassociated apoCaM— Ca²⁺ occupancy of the N lobe leads to CDI, whereas the C lobe mediates CDSA. Alternative splicing of TMEM16A provides a mechanism for regulating apoCaM binding and signaling. TMEM16A splice variants lacking segment a lost apoCaM binding altogether, eliminating both CDSA and CDI, whereas variants specifically lacking segment b were selectively deficient in CDI. Finally, TMEM16A variants defective in apoCaM binding displayed dramatically decreased trafficking to the cell surface.     

Sleep spindles are generated in the absence of T-type calcium channel-mediated low-threshold burst firing of thalamocortical neurons

T-type Ca²⁺ channels in thalamocortical (TC) neurons have long been considered to play a critical role in the genesis of sleep spindles, one of several TC oscillations. A classical model for TC oscillations states that reciprocal interaction between synaptically connected GABAergic thalamic reticular nucleus (TRN) neurons and glutamatergic TC neurons generates oscillations through T-type channel-mediated low-threshold burst firings of neurons in the two nuclei. These oscillations are then transmitted from TC neurons to cortical neurons, contributing to the network of TC oscillations. Unexpectedly, however, we found that both WT and KO mice for Ca_V3.1, the gene for T-type Ca²⁺ channels in TC neurons, exhibit typical waxing-and-waning sleep spindle waves at a similar occurrence and with similar amplitudes and episode durations during non-rapid eye movement sleep. Single-unit recording in parallel with electroencephalography in vivo confirmed a complete lack of burst firing in the mutant TC neurons. Of particular interest, the tonic spike frequency in TC neurons was significantly increased during spindle periods compared with nonspindle periods in both genotypes. In contrast, no significant change in burst firing frequency between spindle and nonspindle periods was noted in the WT mice. Furthermore, spindle-like oscillations were readily generated within intrathalamic circuits composed solely of TRN and TC neurons in vitro in both the KO mutant and WT mice. Our findings call into question the essential role of low-threshold burst firings in TC neurons and suggest that tonic firing is important for the generation and propagation of spindle oscillations in the TC circuit.Sleep spindles are one type of several rhythmic brain waves detected by electroencephalography (EEG) during normal non-rapid eye movement (NREM) sleep. A spindle consists of characteristic waxing-and-waning field potentials grouped into 7- to 14-Hz oscillations that last for 1–3 s and recur once every 5–10 s in the thalamus and the cortex (1–3). Spindles are also visible under anesthesia, particularly with barbiturates but also with ketamine-xylazine combinations (4, 5). These oscillations are generated in the thalamus as a result of synaptic interactions between inhibitory [i.e., thalamic reticular nucleus (TRN)] neurons and excitatory thalamocortical (TC) neurons, and are propagated to the cortex. Corticothalamic projections back to the thalamus complete the cortico-thalamo-cortical loop.In vivo data suggest that TRN neurons are spindle pacemakers, because spindles can be generated in deafferented TRN neurons (6) but disappear in TC regions after disconnection from TRN neurons (7). However, in vitro data suggest that an intact TC-TRN network is a necessity, because spindles are abolished after disconnection of TC and TRN neurons (8).Two distinct firing patterns, tonic and burst, are displayed by both TRN and TC neurons. Burst firing is mediated by low-threshold T-type Ca²⁺ channels (9). Of the three subtypes of T-type channels, Ca_V3.1 is expressed exclusively in TC regions, whereas Ca_V3.2 and Ca_V3.3 are abundant in TRN regions (10). T-type channels in TC neurons have been proposed to be a critical component in the generation of physiological and pathological TC oscillations, such as sleep rhythms (1, 11, 12) and the spike-wave discharges (SWDs) of absence seizures (11, 13, 14). One generally accepted hypothesis proposes that inhibitory inputs from TRN neurons de-inactivate T-type Ca²⁺ channels in TC neurons, leading to induction of burst firings in TC neurons, which in return excite reciprocally connected TRN neurons. These thalamic oscillations are then transmitted from TC neurons to cortical neurons. This model proposes that T-type channel-mediated burst firing in TC neurons underlies sleep spindles and other sleep rhythms within TC circuits (1, 11).There have long been doubts regarding the extent to which TC T-type Ca²⁺ channels contribute to the heterogeneity of TC oscillations during NREM sleep, which consists of multiple EEG components including slow waves (<1 Hz), delta waves (1–4 Hz), and sleep spindles (7–14 Hz). T-type channels have received particular attention in the genesis of spindles and delta waves, both of which are thought to originate from thalamic neurons (8), although cortically generated delta waves also have been found in cats with thalamic lesions (15). A role for TC T-type channels in sleep has been demonstrated in two studies, one using mice with a global deletion (16) and the other using mice with a thalamus-restricted deletion (17) of Ca_V3.1 T-type channels. Both mice exhibited reduced delta waves with intact slow waves (16, 17). Fragmented sleep was observed in both mice, indicating that this sleep phenotype in the global Ca_V3.1^−/− mice is related to a defect in TC neurons. The effect of the mutation on spindle rhythms was unclear, however (16).In the present study, we examined the role of low-threshold burst firing in sleep spindles expressed in TC neurons using mice lacking Ca_V3.1 T-type Ca²⁺ channels. We observed intact sleep spindles in Ca_V3.1^−/− mice during NREM sleep. Our findings suggest that the classical view of the roles of T-type channels and burst firing in TC neurons with respect to the generation of spindle oscillations may need to be revised.     

T-type channel blockade impairs long-term potentiation at the parallel fiber–Purkinje cell synapse and cerebellar learning

Ca_V3.1 T-type channels are abundant at the cerebellar synapse between parallel fibers and Purkinje cells where they contribute to synaptic depolarization. So far, no specific physiological function has been attributed to these channels neither as charge carriers nor more specifically as Ca²⁺ carriers. Here we analyze their incidence on synaptic plasticity, motor behavior, and cerebellar motor learning, comparing WT animals and mice where T-type channel function has been abolished either by gene deletion or by acute pharmacological blockade. At the cellular level, we show that Ca_V3.1 channels are required for long-term potentiation at parallel fiber–Purkinje cell synapses. Moreover, basal simple spike discharge of the Purkinje cell in KO mice is modified. Acute or chronic T-type current blockade results in impaired motor performance in particular when a good body balance is required. Because motor behavior integrates reflexes and past memories of learned behavior, this suggests impaired learning. Indeed, subjecting the KO mice to a vestibulo-ocular reflex phase reversal test reveals impaired cerebellum-dependent motor learning. These data identify a role of low-voltage activated calcium channels in synaptic plasticity and establish a role for Ca_V3.1 channels in cerebellar learning.Neurotransmission at the parallel fiber (PF) and Purkinje cell (PC) synapse plays a pivotal role in cerebellar motor learning probably involving bidirectional changes of its strength (1–3). Unlike in the hippocampus, postsynaptic Ca²⁺ signaling at PF–PC spines may not be dominated by ionotropic glutamatergic receptors, as postsynaptic N-methyl-D-aspartate receptors (NMDARs) are not prominently present at this site and AMPA receptors are predominantly impermeable for calcium ions (4, 5). PCs bear different voltage-dependent Ca channels including P/Q-type (6–8) and T-type channels (9, 10). The spines of PCs contain a high density of Ca_V3.1 T-type channels (11), which can be readily activated by typical bursts of PF activity that occur during sensory stimulation (12–14). To date, the function of the PF to PC synapse plays a pivotal role in cerebellar motor learning, probably involving bidirectional changes of its strength (1–3). Unlike in the hippocampus, T-type channels during PF–PC plasticity induction and cerebellar learning has not been explored.In cerebellar PCs, the elevation of Ca²⁺ in the spine has been suggested to control directly the sign of the changes in synaptic weights (15). Long-term depression (LTD) induction requires conjunctive stimulation of the climbing fibers (CFs) and PFs, which triggers a large supralinear calcium entry mediated by mGluR1, inositol triphosphate (IP3) receptors and voltage-gated calcium channels (16–19). In contrast, long-term potentiation (LTP) develops after PF stimulation only and requires a moderate [Ca²⁺]_i elevation (15). Here, we evaluated the hypothesis that Ca_V3.1 T-type channel activation is essential for LTP and LTP-dependent motor learning.We first looked at PF–PC plasticity of T-type channel blockade/deletion, and then investigated both in vitro and in vivo the dynamics of PC activity as well as the motor behavior of both wild-type and Ca_V3.1 KO mice. Because, in our experiments, motor behavior appears to be impaired in tests requiring a refined body balance, we have analyzed vestibulo-ocular reflex (VOR) adaptation, a learning paradigm more specifically dependent on vestibulo-cerebellar function. We show all three processes to be impaired after T-type channel functional inactivation. We propose that T-type calcium channels contribute to the definition of the learning rules in the cerebellar cortex.