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.     

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels

Facilitation and inactivation of P/Q-type calcium (Ca²⁺) currents through the regulation of voltage-gated Ca²⁺ (Ca_V) 2.1 channels by Ca²⁺ sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express Ca_V2.1 channels. To examine the modulation of endogenous Ca_V2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of Ca_V2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively Ca_V2.1 channels for Ca²⁺ entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10–30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50–100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of Ca_V2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of Ca_V2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.Classic work on the frog neuromuscular junction (NMJ) first described facilitation and depression of synaptic transmission during trains of action potentials (1). These forms of short-term plasticity are widespread among different types of synapses, and they transmit information encoded in the frequency and pattern of action potential generation to postsynaptic cells (2). Calcium (Ca²⁺)-dependent synaptic transmission is mediated by multiple types of voltage-gated Ca²⁺ (Ca_V) channels. Mature mammalian NMJ synapses use exclusively Ca_V2.1 channels to initiate synaptic transmission (3), in contrast to central nervous system synapses that use combinations of Ca_V2.1, Ca_V2.2, and Ca_V2.3 channels (4–6). Disruption of Ca_V2.1 channels by elimination of their pore-forming α1 subunit greatly reduces facilitation at the calyx of Held synapse (7, 8) and the NMJ (9) in mice, suggesting a key role for Ca_V2.1 channels in short-term synaptic plasticity.Ca_V2.1 channels in transfected nonneuronal cells are regulated in a biphasic manner by calmodulin and other related Ca²⁺ sensor (CaS) proteins through interaction with a bipartite regulatory site in their C-terminal domain composed of an IQ-like motif (IM) and a calmodulin-binding domain (CBD) (10–13). CaS proteins interact with the IM motif to initiate Ca²⁺-dependent facilitation in response to local increases in Ca²⁺, and then interact with the CBD to induce Ca²⁺-dependent inactivation in response to longer, more global increases in Ca²⁺ (10–13).The facilitation and inactivation of Ca_V2.1 channels during trains of repetitive stimuli induces synaptic facilitation, followed by a rapid phase of synaptic depression in cultured superior cervical ganglion neurons transiently expressing Ca_V2.1 channels (14). The Ile-Met→Ala-Ala (IM-AA) mutation prevents this synaptic plasticity by altering the interaction of Ca_V2.1 channels with CaS proteins (14). Other CaS proteins can displace calmodulin from their common regulatory site, enhance either facilitation or inactivation of the Ca²⁺ current, and thereby control the direction and amplitude of synaptic plasticity in cultured superior cervical ganglion neurons (15–18). Although previous studies revealed that regulation of Ca_V2.1 channels by CaS proteins can induce and regulate short-term synaptic plasticity in transfected neurons in cell culture, whether this mechanism makes an important contribution to short-term synaptic plasticity in native synapses has remained unknown.To define the functional role of regulation of endogenous Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity in vivo, we introduced the IM-AA mutation into the CaS protein-binding site in the C-terminal domain of Ca_V2.1 channels in mice, and investigated the effects of this mutation on synaptic transmission and short-term synaptic plasticity of NMJ synapses. The IM-AA mutation did not affect basal neuromuscular transmission; however, this mutation blocked short-term synaptic facilitation in response to paired stimuli with short interstimulus intervals (ISIs). Similarly, during high-frequency trains in which the intervals between stimuli are short, the IM-AA mutation reduced synaptic facilitation. In contrast, during trains of stimuli at low frequency with long ISIs, the IM-AA mutation slowed synaptic depression and thereby allowed increased synaptic facilitation. Hindlimb tibialis anterior (TA) muscles of IM-AA mice exhibited reduced peak specific force at 50-Hz stimulation, along with increased muscle fatigue. These defects in muscle function were accompanied by impaired motor control, reduced exercise capacity, and loss of grip strength in IM-AA mice in vivo.Forceful muscle contractions require high-frequency stimulation by the presynaptic motor nerve. Therefore, our results with IM-AA mice link reduced paired-pulse facilitation (PPF) at short ISIs and reduced facilitation during high-frequency trains of stimuli at the cellular level with impaired strength, coordination, and exercise capacity in vivo. These findings demonstrate a critical role for the modulation of Ca_V2.1 channels by CaS proteins in regulating short-term synaptic plasticity, with important consequences for muscle strength and motor control.     

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.     

Conformational signaling required for synaptic plasticity by the NMDA receptor complex

The NMDA receptor (NMDAR) is known to transmit important information by conducting calcium ions. However, some recent studies suggest that activation of NMDARs can trigger synaptic plasticity in the absence of ion flow. Does ligand binding transmit information to signaling molecules that mediate synaptic plasticity? Using Förster resonance energy transfer (FRET) imaging of fluorescently tagged proteins expressed in neurons, conformational signaling is identified within the NMDAR complex that is essential for downstream actions. Ligand binding transiently reduces FRET between the NMDAR cytoplasmic domain (cd) and the associated protein phosphatase 1 (PP1), requiring NMDARcd movement, and persistently reduces FRET between the NMDARcd and calcium/calmodulin-dependent protein kinase II (CaMKII), a process requiring PP1 activity. These studies directly monitor agonist-driven conformational signaling at the NMDAR complex required for synaptic plasticity.Agonist binding to the NMDAR is required for two major forms of synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD) (1). Surprisingly, activation of NMDARs can produce plasticity in opposite directions, with LTP enhancing transmission and LTD reducing transmission. A model was developed (2, 3) to explain how activation of NMDAR could produce these opposing phenomena: strong stimuli during LTP induction drive a large flux of Ca²⁺ through NMDARs, leading to a large increase in intracellular calcium ion concentration ([Ca²⁺]_i) that activates one series of biochemical steps leading to synaptic potentiation; a weaker stimulus during LTD induction drives a more reduced flux of Ca²⁺ through NMDARs, producing a modest increase in [Ca²⁺]_i that activates a different series of biochemical steps, leading to synaptic depression. However, this model is not consistent with recent studies suggesting that no change in [Ca²⁺]_i is required for LTD, and instead invokes metabotropic signaling by the NMDAR (4). Studies supporting an ion-flow-independent role for NMDARs in LTD (4–7) and other processes (7–13) stand in contrast to studies proposing that flow of Ca²⁺ through NMDAR is required for LTD (14) (see ref. 15 for additional references). An important test of an ion-flow-independent model would be to measure directly signaling actions by NMDARs in the absence of ion flow.     

Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700

L-type calcium (Ca²⁺) currents conducted by voltage-gated Ca²⁺ channel Ca_V1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca_V1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca²⁺ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the Ca_V1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca_V1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca²⁺ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.Upon membrane depolarization, Ca_V1.2 channels conduct L-type calcium (Ca²⁺) current into cardiomyocytes and initiate excitation–contraction coupling (1, 2). Ca²⁺ influx through Cav1.2 channels activates Ca²⁺ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation–contraction coupling, Ca²⁺ influx via Ca_V1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca_V1.2 channel (1, 3). Phosphorylation of the Ca_V1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca_V1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4–6). Under those pathological conditions, responsiveness of Ca_V1.2 channel activity to β-adrenergic receptors and PKA activation is severely blunted, resulting in diminished contractile reserve and impaired fight-or-flight response (6, 7). Enormous effort has been devoted to understanding how β-adrenergic regulation of the Ca_V1.2 channel is achieved, but the exact molecular mechanisms remain unresolved.Ca_V1.2 channels contain multiple subunits, including a pore-forming α₁1.2 subunit (also designated α_1C), β and α₂δ subunits that modulate expression of Ca_V1.2 at the cell surface, and possibly γ subunits (8). The closely related Ca_V1.1 and Ca_V1.2 channels in skeletal and cardiac muscle, respectively, are both proteolytically processed near the center of their large C-terminal domains (9, 10), and the distal C terminus (dCT) remains associated noncovalently with the proximal C terminus (pCT) and serves as a potent autoinhibitor (11, 12). Regulation of Ca_V1.2 channels by PKA was reconstituted in nonmuscle cells with a dynamic range of threefold to fourfold similar to native cardiomyocytes by building the autoinhibitory Ca_V1.2 complex through cotransfection of each of its components (13). Successful reconstitution required an A Kinase Anchoring Protein (AKAP), which recruits PKA to the dCT (13–15). Deletion of the dCT in vivo results in loss of regulation of the L-type Ca²⁺ current by the β-adrenergic pathway and embryonic death from heart failure (16, 17). These results suggest that the autoinhibited Ca_V1.2 signaling complex serves as the substrate for β-adrenergic regulation, and disruption of this complex leads to heart failure.PKA is responsible for phosphorylation of the Ca_V1.2 channel in response to β-adrenergic stimulation in cardiac myocytes (18–22). Although multiple PKA sites have been identified in α₁ subunits by in vitro phosphorylation (10, 23), none of these sites is required for regulation of Ca_V1.2 channels in vivo. For example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10, 24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca_V1.2 channel activity in transfected cells or cardiomyocytes (13, 25, 26). Two sites in the C terminus of the skeletal muscle Ca_V1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27). These sites are conserved in cardiac Ca_V1.2 channels as S1700 and T1704, and phosphoproteomics analysis revealed β-adrenergic–stimulated phosphorylation of S1700 by PKA (28). S1700 and T1704 reside at the interface between the pCT and dCT. In studies of the Ca_V1.2 signaling complex reconstituted in nonmuscle cells, phosphorylation of both sites was required for normal basal channel activity, whereas only S1700 was essential for PKA stimulation (13). Mutation of S1700 and T1704 to Ala in STAA mice reduced basal activity and Ca_V1.2 channel regulation by the β-adrenergic pathway in cardiomyocytes (29). To further dissect the contribution of S1700, we studied a mutant mouse line expressing Ca_V1.2 channel with the S1700A mutation (SA mice). Our results demonstrate that this single phosphorylation site is required for normal regulation of Ca_V1.2 channels, contraction of cardiac myocytes, exercise capacity, and cardiac homeostasis.     

Rapid stimulus-evoked astrocyte Ca2+ elevations and hemodynamic responses in mouse somatosensory cortex in vivo

Increased neuron and astrocyte activity triggers increased brain blood flow, but controversy exists over whether stimulation-induced changes in astrocyte activity are rapid and widespread enough to contribute to brain blood flow control. Here, we provide evidence for stimulus-evoked Ca²⁺ elevations with rapid onset and short duration in a large proportion of cortical astrocytes in the adult mouse somatosensory cortex. Our improved detection of the fast Ca²⁺ signals is due to a signal-enhancing analysis of the Ca²⁺ activity. The rapid stimulation-evoked Ca²⁺ increases identified in astrocyte somas, processes, and end-feet preceded local vasodilatation. Fast Ca²⁺ responses in both neurons and astrocytes correlated with synaptic activity, but only the astrocytic responses correlated with the hemodynamic shifts. These data establish that a large proportion of cortical astrocytes have brief Ca²⁺ responses with a rapid onset in vivo, fast enough to initiate hemodynamic responses or influence synaptic activity.Brain function emerges from signaling in and between neurons and associated astrocytes, which causes fluctuations in cerebral blood flow (CBF) (1–5). Astrocytes are ideally situated for controlling activity-dependent increases in CBF because they closely associate with synapses and contact blood vessels with their end-feet (1, 6). Whether or not astrocytic Ca²⁺ responses develop often or rapidly enough to account for vascular signals in vivo is still controversial (7–10). Ca²⁺ responses are of interest because intracellular Ca²⁺ is a key messenger in astrocytic communication and because enzymes that synthesize the vasoactive substances responsible for neurovascular coupling are Ca²⁺-dependent (1, 4). Neuronal activity releases glutamate at synapses and activates metabotropic glutamate receptors on astrocytes, and this activation can be monitored by imaging cytosolic Ca²⁺ changes (11). Astrocytic Ca²⁺ responses are often reported to evolve on a slow (seconds) time scale, which is too slow to account for activity-dependent increases in CBF (8, 10, 12, 13). Furthermore, uncaging of Ca²⁺ in astrocytes triggers vascular responses in brain slices through specific Ca²⁺-dependent pathways with a protracted time course (14, 15). More recently, stimulation of single presynaptic neurons in hippocampal slices was shown to evoke fast, brief, local Ca²⁺ elevations in astrocytic processes that were essential for local synaptic functioning in the adult brain (16, 17). This work prompted us to reexamine the characteristics of fast, brief astrocytic Ca²⁺ signals in vivo with special regard to neurovascular coupling, i.e., the association between local increases in neural activity and the concomitant rise in local blood flow, which constitutes the physiological basis for functional neuroimaging.Here, we describe how a previously undescribed method of analysis enabled us to provide evidence for fast Ca²⁺ responses in a main fraction of astrocytes in mouse whisker barrel cortical layers II/III in response to somatosensory stimulation. The astrocytic Ca²⁺ responses were brief enough to be a direct consequence of synaptic excitation and correlated with stimulation-induced hemodynamic responses. Fast Ca²⁺ responses in astrocyte end-feet preceded the onset of dilatation in adjacent vessels by hundreds of milliseconds. This finding might suggest that communication at the gliovascular interface contributes considerably to neurovascular coupling.     

Pathophysiological implication of CaV3.1 T-type Ca2+ channels in trigeminal neuropathic pain

A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in Ca_V3.1 T-type Ca²⁺ channel knockout and wild-type mice. Ca_V3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in Ca_V3.1 knockout mice. Our results suggest that the presence of Ca_V3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.Since 1911, when H. Head and G. M. Holmes first addressed the relevance of the thalamus as a central pattern generator for neuropathic pain (1), many clinical studies have indicated the coexistence of pathophysiological thalamocortical activity and the occurrence of neuropathic pain. Compared with healthy controls, patients with neuropathic pain show increased low-frequency thalamocortical oscillations in magnetoencephalogram (MEG) recordings. Such low-frequency oscillations are a typical thalamocortical dysrhythmia (TCD) syndrome (2). In agreement with such MEG findings, the excess power of low-frequency oscillation was marked in local-field potential (LFP) recordings from the thalamus (3–5) and electroencephalogram (EEG) recordings from the cortex (6, 7) of patients with neuropathic pain. In addition, the presence of thalamic burst firing, which is a well-known underlying mechanism for cortical low-frequency oscillations through the thalamocortical recurrent network (8, 9), has been confirmed in patients with neuropathic pain (10–13). Results from small lesions in the posterior part of the central lateral nucleus of the medial thalamus, which reduce tonic hyperpolarization of thalamic neurons in chronic neuropathic pain patients, have also provided insight into the role of low-frequency thalamic rhythmicity in neuropathic pain. Following such interventions, a marked decrease in low-frequency EEG power was observed as well as pain relief (7, 12), indicating that alteration of thalamocortical rhythms plays a crucial role in the development and/or persistence of neuropathic pain.Trigeminal neuropathic pain (TNP) is characterized by unilateral chronic facial pain limited to one or more divisions of the trigeminal nerve. There is increasing evidence that TNP is associated with anatomical and biochemical changes in the thalamus (14–16). Moreover, patients with TNP display significant reductions in thalamic volume and neural viability (15), indicating that altered thalamic anatomy, physiology, and biochemistry may result in disturbed thalamocortical oscillatory properties.Abnormal thalamic activity has been investigated in patients with neuropathic pain (3–7, 10–13, 17, 18), including TNP (14–16). Furthermore, the potential role of thalamic burst firing in abnormally increased low-frequency oscillations has been proposed as a pathophysiological mechanism (2, 12). Because T-type Ca²⁺ channels are known to underlie thalamic burst firing (8, 9), it is reasonable to propose that pathophysiological low-frequency rhythms, such as those seen in central neuropathic pain, may be mediated by these calcium channels. Nevertheless, this hypothesis has not been directly tested. To determine whether T-type Ca²⁺ channels play a role in the generation of neuropathic pain, thalamocortical oscillatory properties were examined in mice lacking Ca_V3.1 channels following induction of TNP through partial ligation of the inferior orbital nerve (IoN). This channel represents the major T-type Ca²⁺ channel isoform in thalamocortical projection neurons (19). Following IoN ligations, Ca_V3.1 knockout (KO) mice showed significantly attenuated mechanical hypersensitivity, compared with wild-type (WT) mice. Moreover, spectral analysis of thalamocortical rhythms from Ca_V3.1 KO mice showed decreased low-frequency rhythm propensity, compared with WT mice. In addition, response to gamma activation and the spatiotemporal patterns of primary somatosensory (S1) cortex activity were altered in WT but not in KO mice after IoN ligation. Moreover, the cross-frequency interactions between low-frequency and gamma rhythms were significantly increased in WT but not in Ca_V3.1 KO mice. These findings indicate that TNP is associated with altered thalamocortical rhythms, resulting in increased sensitivity to pain as well as pain generation in response to nonnoxious stimuli. In addition, these results indicate that Ca_V3.1 T-type Ca²⁺ channels are fundamentally associated with the alteration of thalamocortical rhythms seen in TNP.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses

The tight spatial coupling of synaptic vesicles and voltage-gated Ca²⁺ channels (Ca_Vs) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca²⁺ channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission. We investigated RIM-BP2–deficient murine hippocampal neurons in cultures and slices. Short-term facilitation is significantly enhanced in both model systems. Detailed analysis in culture revealed a reduction in initial release probability, which presumably underlies the increased short-term facilitation. Superresolution microscopy revealed an impairment in Ca_V2.1 clustering at AZs, which likely alters Ca²⁺ nanodomains at release sites and thereby affects release probability. Additional deletion of RIM-BP1 does not exacerbate the phenotype, indicating that RIM-BP2 is the dominating RIM-BP isoform at these synapses.At the presynapse, coupling between action potentials (APs) and synaptic vesicle fusion is exquisitely precise, ensuring high temporal fidelity of neuron-to-neuron signaling in the nervous system. Two properties are thought to be responsible for this remarkable precision: a highly efficient release apparatus that transduces Ca²⁺ signals into vesicle fusion and a tightly organized active zone (AZ), where the release apparatus and voltage-gated Ca²⁺ channels (Ca_Vs) are spatially coupled. Rab-interacting molecules (RIM) are thought to contribute to both properties, because loss of RIM impairs vesicle priming (1) and Ca_V localization at the AZ (2). RIM-binding proteins (RIM-BPs) directly interact with RIM (3), the pore-forming subunits of Ca_V1 and Ca_V2 channels (2, 4, 5), and Bassoon (5), and have therefore been suggested to play a role in presynaptic Ca_V localization. The Drosophila homolog of RIM-binding proteins (DRBP) is indeed crucial for neurotransmitter release at the AZ of neuromuscular junctions (NMJs) because loss of DRBP reduces Ca_V abundance and impairs the integrity of the AZ scaffold (6). DRBP-deficient flies show severe impairment of neurotransmitter release along with increased short-term facilitation (6, 7).Recently, Acuna et al. (8) published a report on the combined loss of RIM-BP1 and RIM-BP2 in mouse synapses. The authors report that although RIM-BPs are not essential for synaptic transmission, AP-triggered neurotransmitter release is more variable and the sensitivity to the Ca²⁺ chelator EGTA is increased at the Calyx of Held, suggesting a larger coupling distance of Ca_V and the release machinery.In the present study, we further investigated the consequences of constitutive deletion of RIM-BP2 on the structure and function of mouse hippocampal synapses. We show that loss of RIM-BP2 leads to a moderate reduction in initial release probability, which translates into profound changes in short-term plasticity (STP). This deficit can be overcome by increasing extracellular Ca²⁺. We established triple-channel time-gated stimulated emission depletion (gSTED) microscopy for RIM-BP2, Munc13-1, and Bassoon, as well as for Ca_V2.1, RIM, and the postsynaptic marker protein Homer1. Using this technique, we demonstrate that although synapse number and molecular architecture appear essentially intact, RIM-BP2 is necessary for proper coclustering of the P/Q-type Ca_V subunit Ca_V2.1 with the AZ protein Bassoon at hippocampal CA3-CA1 synapses. We hypothesize that the observed change in Ca_V localization causes a discrete alteration in the coupling of Ca²⁺ influx and exocytosis, and thereby modifies release probability and, consequently, STP. Additional deletion of RIM-BP1 did not strengthen the changes in short-term facilitation, supporting our hypothesis that RIM-BP2 is the major RIM-BP paralog at glutamatergic hippocampal synapses.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.