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.     

Altered pain responses in mice lacking alpha 1E subunit of the voltage-dependent Ca2+ channel

alpha(1) subunit of the voltage-dependent Ca(2+) channel is essential for channel function and determines the functional specificity of various channel types. alpha(1E) subunit was originally identified as a neuron-specific one, but the physiological function of the Ca(2+) channel containing this subunit (alpha(1E) Ca(2+) channel) was not clear compared with other types of Ca(2+) channels because of the limited availability of specific blockers. To clarify the physiological roles of the alpha(1E) Ca(2+) channel, we have generated alpha(1E) mutant (alpha(1E)-/-) mice by gene targeting. The lacZ gene was inserted in-frame and used as a marker for alpha(1E) subunit expression. alpha(1E)-/- mice showed reduced spontaneous locomotor activities and signs of timidness, but other general behaviors were apparently normal. As involvement of alpha(1E) in pain transmission was suggested by localization analyses with 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside staining, we conducted several pain-related behavioral tests using the mutant mice. Although alpha(1E)+/- and alpha(1E)-/- mice exhibited normal pain behaviors against acute mechanical, thermal, and chemical stimuli, they both showed reduced responses to somatic inflammatory pain. alpha(1E)+/- mice showed reduced response to visceral inflammatory pain, whereas alpha(1E)-/- mice showed apparently normal response compared with that of wild-type mice. Furthermore, alpha(1E)-/- mice that had been presensitized with a visceral noxious conditioning stimulus showed increased responses to a somatic inflammatory pain, in marked contrast with the wild-type mice in which long-lasting effects of descending antinociceptive pathway were predominant. These results suggest that the alpha(1E) Ca(2 +) channel controls pain behaviors by both spinal and supraspinal mechanisms.     

Asynchronous Ca2+ current conducted by voltage-gated Ca2+ (CaV)-2.1 and CaV2.2 channels and its implications for asynchronous neurotransmitter release

We have identified an asynchronously activated Ca(2+) current through voltage-gated Ca(2+) (Ca(V))-2.1 and Ca(V)2.2 channels, which conduct P/Q- and N-type Ca(2+) currents that initiate neurotransmitter release. In nonneuronal cells expressing Ca(V)2.1 or Ca(V)2.2 channels and in hippocampal neurons, prolonged Ca(2+) entry activates a Ca(2+) current, I(Async), which is observed on repolarization and decays slowly with a half-time of 150-300 ms. I(Async) is not observed after L-type Ca(2+) currents of similar size conducted by Ca(V)1.2 channels. I(Async) is Ca(2+)-selective, and it is unaffected by changes in Na(+), K(+), Cl(-), or H(+) or by inhibitors of a broad range of ion channels. During trains of repetitive depolarizations, I(Async) increases in a pulse-wise manner, providing Ca(2+) entry that persists between depolarizations. In single-cultured hippocampal neurons, trains of depolarizations evoke excitatory postsynaptic currents that show facilitation followed by depression accompanied by asynchronous postsynaptic currents that increase steadily during the train in parallel with I(Async). I(Async) is much larger for slowly inactivating Ca(V)2.1 channels containing β(2a)-subunits than for rapidly inactivating channels containing β(1b)-subunits. I(Async) requires global rises in intracellular Ca(2+), because it is blocked when Ca(2+) is chelated by 10 mM EGTA in the patch pipette. Neither mutations that prevent Ca(2+) binding to calmodulin nor mutations that prevent calmodulin regulation of Ca(V)2.1 block I(Async). The rise of I(Async) during trains of stimuli, its decay after repolarization, its dependence on global increases of Ca(2+), and its enhancement by β(2a)-subunits all resemble asynchronous release, suggesting that I(Async) is a Ca(2+) source for asynchronous neurotransmission.     

Multiple Ca2+ channel types coexist to regulate synaptosomal neurotransmitter release.

The regulation of excitation-secretion coupling by Ca2+ channels is a fundamental property of the nerve terminal. Peptide toxins that block specific Ca2+ channel types have been used to identify which channels participate in neurotransmitter release. Subsecond measurements of [3H]-glutamate and [3H]dopamine release from rat striatal synaptosomes showed that P-type channels, which are sensitive to the Agelenopsis aperta venom peptide omega-Aga-IVA, trigger the release of both transmitters. Dopamine (but not glutamate) release was also controlled by N-type, omega-conotoxin-sensitive channels. With strong depolarizations, where neither toxin was very effective alone, a combination of omega-Aga-IVA and omega-conotoxin produced a synergistic inhibition of 60-80% of Ca(2+)-dependent dopamine release. The results suggest that multiple Ca2+ channel types coexist to regulate neurosecretion under normal physiological conditions in the majority of nerve terminals. P- and N-type channels coexist in dopaminergic terminals, while P-type and a omega-conotoxin- and omega-Aga-IVA-resistant channel coexist in glutamatergic terminals. Such an arrangement could lend a high degree of flexibility in the regulation of transmitter release under diverse conditions of stimulation and modulation.     

Postsynaptic P/Q-type Ca2+ channel in Purkinje cell mediates synaptic competition and elimination in developing cerebellum

Neural circuits are initially redundant but rearranged through activity-dependent synapse elimination during postnatal development. This process is crucial for shaping mature neural circuits and for proper brain function. At birth, Purkinje cells (PCs) in the cerebellum are innervated by multiple climbing fibers (CFs) with similar synaptic strengths. During postnatal development, a single CF is selectively strengthened in each PC through synaptic competition, the strengthened single CF undergoes translocation to a PC dendrite, and massive elimination of redundant CF synapses follows. To investigate the cellular mechanisms of this activity-dependent synaptic refinement, we generated mice with PC-selective deletion of the Ca(v)2.1 P/Q-type Ca(2+) channel, the major voltage-dependent Ca(2+) channel in PCs. In the PC-selective Ca(v)2.1 knockout mice, Ca(2+) transients induced by spontaneous CF inputs are markedly reduced in PCs in vivo. Not a single but multiple CFs were equally strengthened in each PC from postnatal day 5 (P5) to P8, multiple CFs underwent translocation to PC dendrites, and subsequent synapse elimination until around P12 was severely impaired. Thus, P/Q-type Ca(2+) channels in postsynaptic PCs mediate synaptic competition among multiple CFs and trigger synapse elimination in developing cerebellum.     

Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1

Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.     

Ablation of P/Q-type Ca(2+) channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the alpha(1A)-subunit

The Ca(2+) channel alpha(1A)-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca(2+) channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. alpha(1A)-Subunits are thought to support both P- and Q-type Ca(2+) channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca(2+) delivery system at excitatory nerve terminals. We generated alpha(1A)-deficient mice (alpha(1A)(-/-)) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying approximately 3-4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca(2+) channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in alpha(1A)(-/-) hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca(2+) entry. The alpha(1A)(-/-) mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in alpha(1A).     

Ca2+ channels, 'quantized' Ca2+ release, and differentiation of myocytes in the cardiovascular system

The application of confocal microscopy to cardiac and skeletal muscle has resulted in the observation of transient, spatially localized elevations in [Ca2+]i, termed 'Ca2+ sparks'. Ca2+ sparks are thought to represent 'elementary' Ca2+ release events, which arise from one or more ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. In cardiac muscle, Ca2+ sparks appear to be key elements of excitation-contraction coupling, in which the global [Ca2+]i transient is thought to involve the recruitment of Ca2+ sparks, each of which is controlled locally by single coassociated L-type Ca2+ channels. Recently, Ca2+ sparks have been detected in smooth muscle cells of arteries. In this review, we analyse the complex relationship of Ca2+ influx and Ca2+ release with local, subcellular Ca2+ microdomains in light of recent studies on Ca2+ sparks in cardiovascular cells. We performed a comparative analysis of 'elementary' Ca2+ release units in mouse, rat and human arterial smooth muscle cells, using measurements of Ca2+ sparks and plasmalemmal K(Ca) currents activated by Ca2+ sparks (STOCs). Furthermore, the appearance of Ca2+ sparks during ontogeny of arterial smooth muscle is explored. Using intact pressurized arteries, we have investigated whether RyRs causing Ca2+ sparks (but not smaller 'quantized' Ca2+ release events, e.g. hypothetical 'Ca2+ quarks') function as key signals that, through membrane potential and global cytoplasmic [Ca2+], oppose arterial myogenic tone and influence vasorelaxation. We believe that voltage-dependent Ca2+ channels and local RyR-related Ca2+ signals are important in differentiation, proliferation, and gene expression. Our findings suggest that 'elementary' Ca2+ release units may represent novel potent therapeutic targets for regulating function of intact arterial smooth muscle tissue.     

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.     

超压力负荷诱导肥厚心肌的肌浆网钙摄取和释放的改变

目的探讨超压力负荷下左心室心肌肌浆网钙ATP酶、雷诺定受体2(ryanodine receptor2,RYR2)和三磷酸肌醇受体1(inositol1,4,5-trisphosphate receptor1,IP3R1)变化以及血管紧张素Ⅱ受体阻断药的影响。方法用腹主动脉缩窄法建立大鼠超压力负荷模型。检测心肌肌浆网钙ATP酶活性、Ca2 最大摄取速率、Ca2 最大摄取量、3H-雷诺定与RYR2最大结合量和RYR2受体密度。用免疫印迹法检测心肌肌浆网钙ATP酶2a(SERCA2a)蛋白表达;用反转录-聚合酶链反应检测心肌RYR2和IP3R1的mRNA表达。结果高压力负荷下左心室心肌呈典型的肥厚心肌的形态改变。手术组左心室心肌内浆网钙ATP酶活性、Ca2 最大摄取速度、Ca2 摄取量、RYR2最大结合量、RYR2的mRNA表达[吸光度/磷酸甘油醛脱氢酶]、IP3R1mRNA表达[吸光度/磷酸甘油醛脱氢酶]均低于假手术组(差异有统计学意义,P<0.01,n=12),缬沙坦组高于手术组及PD123319组(P<0.05),手术组与PD123319组间差异无统计学意义(P>0.05,n=12)。结论超压力负荷诱导的肥厚心肌组织钙调节能力明显下降,但缬沙坦可改善肥厚心肌组织的钙调节能力。     

Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca2+ channel activity and inhibitory synaptic transmission

Inhibitory synapses play key roles in the modulatory circuitry that regulates pain signaling and generation of migraine headache. A rare, dominant form of this common disease, familial hemiplegic migraine type 1 (FHM1), arises from missense mutations in the pore-forming alpha1A subunit of P/Q-type Ca2+ channels. These channels are normally vital for presynaptic Ca2+ entry and neurotransmitter release at many central synapses, raising questions about effects of FHM1 mutations on neuronal Ca2+ influx and inhibitory and excitatory neurotransmission. We have expressed the four original FHM1 mutant channels in hippocampal neurons from alpha1A knockout mice. Whole-cell recordings indicated that FHM1 mutant channels were less effective than wild-type channels in their ability to conduct P/Q-type current, but not generally different from wild type in voltage-dependent channel gating. Ca2+ influx triggered by action potential waveforms was also diminished. In keeping with decreased channel activity, FHM1 mutant channels were correspondingly impaired in supporting the P/Q-type component of inhibitory neurotransmission. When expressed in wild-type inhibitory neurons, FHM1 mutant channels reduced the contribution of P/Q-type channels to GABAergic synaptic currents, consistent with a competition of mutant and endogenous channels for P/Q-specific slots. In all cases, N-type channels took up the burden of supporting transmission and homeostatic mechanisms maintained overall synaptic strength. The shift to reliance on N-type channels greatly increased the susceptibility to G protein-coupled modulation of neurotransmission, studied with the GABAB agonist baclofen. Thus, mutant-expressing synapses might be weakened in a heightened state of neuromodulation like that provoked by triggers of migraine such as stress.     

Pharmacological basis of Ca2+ channels and Ca2+ channel antagonists

Ca2+ plays multiple roles in muscle E-C coupling, secretion, and neural transmission, in addition to survival, proliferation, and death of cells. The voltage-dependent L-type Ca2+ channel is a transmembrane protein that selectively permeates Ca2+ on activation by membrane depolarization. Ca2+ channel blockers (or Ca2+ antagonists) selectively block this channel. The blocking action is exerted in a tissue-specific manner, which underlies the unique pharmacological properties of Ca2+ channel blockers. The later generation of slowly-acting and long-lasting Ca2+ channel blockers has been designed to overcome the side effects of classical Ca2+ channel blockers. The pharmacological and molecular basis for the unique action of Ca2+ channel blockers will be discussed.     

Functional uncoupling between Ca2+ release and afterhyperpolarization in mutant hippocampal neurons lacking junctophilins

Junctional membrane complexes (JMCs) composed of the plasma membrane and endoplasmic/sarcoplasmic reticulum seem to be a structural platform for channel crosstalk. Junctophilins (JPs) contribute to JMC formation by spanning the sarcoplasmic reticulum membrane and binding with the plasma membrane in muscle cells. In this article, we report that mutant JP double-knockout (JP-DKO) mice lacking neural JP subtypes exhibited an irregular hindlimb reflex and impaired memory. Electrophysiological experiments indicated that the activation of small-conductance Ca(2+)-activated K(+) channels responsible for afterhyperpolarization in hippocampal neurons requires endoplasmic reticulum Ca(2+) release through ryanodine receptors, triggered by NMDA receptor-mediated Ca(2+) influx. We propose that in JP-DKO neurons lacking afterhyperpolarization, the functional communications between NMDA receptors, ryanodine receptors, and small-conductance Ca(2+)-activated K(+) channels are disconnected because of JMC disassembly. Moreover, JP-DKO neurons showed an impaired long-term potentiation and hyperactivation of Ca(2+)/calmodulin-dependent protein kinase II. Therefore, JPs seem to have an essential role in neural excitability fundamental to plasticity and integrated functions.     

Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C2 domains mediate neurotransmitter release

Neurotransmitter release is triggered by cooperative Ca²⁺-binding to the Ca²⁺-sensor protein synaptotagmin-1. Synaptotagmin-1 contains two C2 domains, referred to as the C2A and C2B domains, that bind Ca²⁺ with similar properties and affinities. However, Ca²⁺ binding to the C2A domain is not required for release, whereas Ca²⁺ binding to the C2B domain is essential for release. We now demonstrate that despite its expendability, Ca²⁺-binding to the C2A domain significantly contributes to the overall triggering of neurotransmitter release, and determines its Ca²⁺ cooperativity. Biochemically, Ca²⁺ induces more tight binding of the isolated C2A domain than of the isolated C2B domain to standard liposomes composed of phosphatidylcholine and phosphatidylserine. However, here we show that surprisingly, the opposite holds true when the double C2A/B-domain fragment of synaptotagmin-1 is used instead of isolated C2 domains, and when liposomes containing a physiological lipid composition are used. Under these conditions, Ca²⁺ binding to the C2B domain but not the C2A domain becomes the primary determinant of phospholipid binding. Thus, the unique requirement for Ca²⁺ binding to the C2B domain for synaptotagmin-1 in Ca²⁺-triggered neurotransmitter release may be accounted for, at least in part, by the unusual phospholipid-binding properties of its double C2A/B-domain fragment.     

Clustering of Ca2+ channels and Ca(2+)-activated K+ channels at fluorescently labeled presynaptic active zones of hair cells.

Electrical resonance, which in some hair cells provides a mechanism for frequency tuning, is mediated by clusters of Ca2+ channels and Ca(2+)-activated K+ channels that have been proposed to occur at presynaptic active zones. To localize Ca2+ channels on the cellular surface, we loaded hair cells from the frog's sacculus with the Ca2+ indicator fluo-3 and imaged them by fluorescence confocal microscopy. When a cell was depolarized, we observed on its basolateral surface several foci of transiently enhanced fluorescence due to local Ca2+ influx. After protracted recording, each cell displayed on average 18 brightly and permanently fluorescent spots at the same positions. We mapped these spots in four hair cells and compared their locations with those of presynaptic active zones, as determined from transmission electron micrographs of serial sections through the same cells. The results demonstrated that enhanced fluo-3 fluorescence marks active zones. Measurement of currents through membrane patches at fluorescently labeled active zones demonstrated that both voltage-activated Ca2+ channels and Ca(2+)-activated K+ channels occur there. These results confirm that the ion channels involved in electrical tuning and synaptic transmission by hair cells cluster together at presynaptic active zones.     

Autocrine activation of P2Y1 receptors couples Ca2+ influx to Ca2+ release in human pancreatic beta cells

Aims/hypothesis

There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells.

Methods

We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion in human beta cells.

Results

Extracellular application of ATP evoked small inward currents (3.4?±?0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4?±?2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca²⁺]_i by stimulating Ca²⁺ influx and evoking Ca²⁺ release via InsP₃-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca²⁺ release was sufficient to trigger exocytosis in cells voltage-clamped at ?70 mV. All effects of ATP were mimicked by the P2Y_(1/12/13) agonist ADP and the P2Y₁ agonist MRS-2365, whereas the P2X_(1/3) agonist α,β-methyleneadenosine-5-triphosphate only had a small effect. The P2Y₁ antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca²⁺]_i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca²⁺]_i and exocytosis without affecting Ca²⁺ influx.

Conclusions/interpretation

These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y₁ receptors, stimulating electrical activity and coupling Ca²⁺ influx to Ca²⁺ release from ER stores.     

Structure and function of Ca2+ channels

Ca(2+) influx across plasma membrane is critical to evoke increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that controls various physiological and pathological responses. Recently, diverse Ca(2+) channels responsible for Ca(2+) influx have been identified. In this review, we attempt to overview molecular entities of Ca(2+) channels, and their structure, function, and biological roles.     

Functional importance of L- and P/Q-type voltage-gated calcium channels in human renal vasculature

Calcium channel blockers are widely used for treatment of hypertension, because they decrease peripheral vascular resistance through inhibition of voltage-gated calcium channels. Animal studies of renal vasculature have shown expression of several types of calcium channels that are involved in kidney function. It was hypothesized that human renal vascular excitation-contraction coupling involves different subtypes of channels. In human renal artery and dissected intrarenal blood vessels from nephrectomies, PCR analysis showed expression of L-type (Ca(v) 1.2), P/Q-type (Ca(v) 2.1), and T-type subtype (Ca(v) 3.1 and Ca(v) 3.2) voltage-gated calcium channels (Ca(v)s), and quantitative PCR showed highest expression of L-type channels in renal arteries and variable expression between patients of subtypes of calcium channels in intrarenal vessels. Immunohistochemical labeling of kidney sections revealed signals for Ca(v) 2.1 and Ca(v) 3.1 associated with smooth muscle cells of preglomerular and postglomerular vessels. In human intrarenal arteries, depolarization with potassium induced a contraction inhibited by the L-type antagonist nifedipine, EC(50) 1.2×10(-8) mol/L. The T-type antagonist mibefradil inhibited the potassium-induced constriction with large variations between patients. Interestingly, the P/Q-type antagonist, ω-agatoxin IVA, inhibited significantly the contraction with 24% at 10(-9) mol/L. In conclusion L-, P/Q, and T-type channels are expressed in human renal blood vessels, and L- and P/Q-type channels are of functional importance for the depolarization-induced vasoconstriction. The contribution of P/Q-type channels to contraction in the human vasculature is a novel mechanism for the regulation of renal blood flow and suggests that clinical treatment with calcium blockers might affect vascular reactivity also through P/Q-type channel inhibition.     

Luminal Ca2+ controls termination and refractory behavior of Ca2+-induced Ca2+ release in cardiac myocytes

Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivation of the SR Ca2+ release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+ regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+ buffering on global and local Ca2+ release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+ buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to "internal" solutions containing the buffers. Enhanced Ca2+ buffering in the SR was confirmed by an increase in the total SR Ca2+ content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+ transients induced by I(Ca) and deranged the smoothly graded I(Ca)-SR Ca2+ release relationship. The amplitude and time-to-peak of local Ca2+ release events, Ca2+ sparks, as well as the duration of local Ca2+ release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+ buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+ release sites in cardiac muscle.     

Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates arterial tone and blood pressure

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca2+-activated K+ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure- and phenylephrine-induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure.