Single Calcium Channels Research Articles

Association between Hypertrophic Cardiomyopathy and Variations in Sarcomere Gene and Calcium Channel Gene in Adults

Introduction: Calcium channel gene variations have been reported to be associated with hypertrophic cardiomyopathy (HCM) in family, but the relationship between calcium channel gene variations and HCM remains undefined in the population. Methods: A total of 719 HCM unrelated patients were initially enrolled. Finally, 371 patients were identified based on inclusion and exclusion criteria, including 145 patients with gene negative, 28 patients with a single rare calcium channel gene variation (calcium gene variation), 162 patients with a single pathogenic/likely pathogenic sarcomere gene variation (sarcomere gene variation) and 36 patients with a single pathogenic/likely pathogenic sarcomere gene variation and a single rare calcium channel gene variation (double gene variations). Then the demographic, electrocardiographic, echocardiographic, and follow-up data were collected. Results: Patients with double gene variations were at an earlier age and had more percent of family history of HCM, and had thicker walls, higher left ventricular outflow tract pressure gradient, more pathological Q waves, and more bundle branch blocks as compared with those with single sarcomere gene variation. During the follow-up period, patients with double gene variations had more primary endpoints than the other three groups (p = 0.0013). Multivariate analysis showed that double gene variations were the independent predictor of primary endpoint events in patients (HR: 4.82, 95% CI: 1.77–13.2; p = 0.002). Conclusion: We found that patients with double gene variations had more severe HCM phenotype and prognosis. The pathogenesis effects of sarcomere gene variation and calcium channel gene variation may be cumulative in HCM populations.

Magnesium Ions Moderate Calcium-Induced Calcium Release in Cardiac Calcium Release Sites by Binding to Ryanodine Receptor Activation and Inhibition Sites.

Ryanodine receptor channels at calcium release sites of cardiac myocytes operate on the principle of calcium-induced calcium release. In vitro experiments revealed competition of Ca2+ and Mg2+ in the activation of ryanodine receptors (RyRs) as well as inhibition of RyRs by Mg2+. The impact of RyR modulation by Mg2+ on calcium release is not well understood due to the technical limitations of in situ experiments. We turned instead to an in silico model of a calcium release site (CRS), based on a homotetrameric model of RyR gating with kinetic parameters determined from in vitro measurements. We inspected changes in the activity of the CRS model in response to a random opening of one of 20 realistically distributed RyRs, arising from Ca2+/Mg2+ interactions at RyR channels. Calcium release events (CREs) were simulated at a range of Mg2+-binding parameters at near-physiological Mg2+ and ATP concentrations. Facilitation of Mg2+ binding to the RyR activation site inhibited the formation of sparks and slowed down their activation. Impeding Mg-binding to the RyR activation site enhanced spark formation and speeded up their activation. Varying Mg2+ binding to the RyR inhibition site also dramatically affected calcium release events. Facilitation of Mg2+ binding to the RyR inhibition site reduced the amplitude, relative occurrence, and the time-to-end of sparks, and vice versa. The characteristics of CREs correlated dose-dependently with the effective coupling strength between RyRs, defined as a function of RyR vicinity, single-channel calcium current, and Mg-binding parameters of the RyR channels. These findings postulate the role of Mg2+ in calcium release as a negative modulator of the coupling strength among RyRs in a CRS, translating to damping of the positive feedback of the calcium-induced calcium-release mechanism.

Systematic review and Meta-analysis on efficacy and safety of Yangxue Qingnao Granules in treatment of migraine

To evaluate the efficacy and safety of Yangxue Qingnao Granules alone or combined with calcium channel blocker in treatment of migraine. In this study, four Chinese databases(CNKI, VIP, WanFang, CBM), three English databases(Cochrane Library, EMbase, Medline) and clinical trials registration center(ClinicalTrials.gov) were retrieved. The retrieval time was from the establishment of each database to January 8, 2020. According to the set inclusion criteria and exclusion criteria,the randomized controlled trial(RCT) of Yangxue Qingnao Granules alone or combined with calcium channel blocker was selected. The "Cochrane bias risk assessment" tool was used to evaluate the quality of the included studies. RevMan 5.3 was used to conduct Meta-analysis of the included studies and grade system was used to evaluate the evidence quality of the outcome indicators. A total of 583 documents were retrieved and finally included in 23 studies, with a total sample size of 2 308 cases, 1 171 cases in the treatment group and 1 137 cases in the control group. The overall quality of the research included was not high. Meta-analysis showed that,(1)in terms of effective rate, Yangxue Qingnao Granules combined with calcium channel blocker was better than calcium channel blocker(RR=1.24, 95%CI[1.17, 1.32], P<0.000 01), and there was no significant difference between Yangxue Qingnao Granules and calcium channel blocker(RR=1.36, 95%CI[0.91, 2.03], P=0.14).(2)In terms of reducing headache frequency, when the unit of headache frequency was times per month, Yangxue Qingnao Granules combined with calcium channel blocker was better than calcium channel blocker(MD=-1.39, 95%CI[-1.83,-0.95], P<0.000 01), when the unit of headache frequency was times daily, Yangxue Qingnao Granules combined with calcium channel blocker was better than calcium channel blocker(MD=-2.08, 95%CI[-2.34,-1.82], P<0.000 01).(3)In terms of headache intensity, when headache intensity was scored by pain intensity, Yangxue Qingnao Granules combined with calcium channel blocker was better than calcium channel blocker(MD=-0.70, 95%CI[-0.81,-0.59], P<0.000 01), when headache intensity was scored by VAS score, Yangxue Qingnao Granules combined with calcium channel blocker was better than calcium channel blocker(MD=-1.59, 95%CI[-2.13,-1.06], P<0.000 01).(4)In terms of reducing headache duration, Yangxue Qingnao Granules combined with calcium channel blocker was better than calcium channel blocker(SMD=-3.13, 95%CI[-4.12,-2.15], P<0.000 01). GRADE system showed that the evidence level of the above outcome indicators was low and extremely low. Twelve cases of adverse reactions were reported, all of which were mild. The results showed that the combination of Yang-xue Qingnao Granules can improve the effective rate, reduce the headache frequency, the headache intensity and the headache duration, and had good safety and low incidence of adverse reactions compared with the single calcium channel blocker. However, there was no difference in the effective rate between Yangxue Qingnao Granules alone and calcium channel blocker. In view of the low quality of this study, which affects the reliability of the conclusion, it is necessary to use the conclusion of this study carefully, and more high-quality randomized controlled trials are needed to further verify in the future.

Electrophysiological Features of Single Store-Operated Calcium Channels in HEK S4 Cell Line with Stable STIM1 Protein Knockdown

An important role in intracellular calcium signaling is played by store-operated channels activated by STIM proteins, calcium sensors of the endoplasmic reticulum. In stable STIM1 knockdown HEK S4 cells, single channels activated by depletion of intracellular calcium stores were detected by cell-attached patch-clamp technique and their electrophysiological parameters were described. Comparison of the properties of single channels in HEK293 and HEK S4 cells revealed no significant differences in their current-voltage curves, while regulation of store-operated calcium channels in these cell lines depended on the level of STIM1 expression. We can conclude that electrophysiological peculiarities of store-regulated calcium entry observed in different cells can be explained by differences in STIM1 expression.

Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue.

Four-domain voltage-gated Ca2+ (Cav) channels play fundamental roles in the nervous system, but little is known about when or how their unique properties and cellular roles evolved. Of the three types of metazoan Cav channels, Cav1 (L-type), Cav2 (P/Q-, N- and R-type) and Cav3 (T-type), Cav3 channels are optimized for regulating cellular excitability because of their fast kinetics and low activation voltages. These same properties permit Cav3 channels to drive low-threshold exocytosis in select neurons and neurosecretory cells. Here, we characterize the single T-type calcium channel from Trichoplax adhaerens (TCav3), an early diverging animal that lacks muscle, neurons, and synapses. Co-immunolocalization using antibodies against TCav3 and neurosecretory cell marker complexin labeled gland cells, which are hypothesized to play roles in paracrine signaling. Cloning and in vitro expression of TCav3 reveals that, despite roughly 600 million years of divergence from other T-type channels, it bears the defining structural and biophysical features of the Cav3 family. We also characterize the channel's cation permeation properties and find that its pore is less selective for Ca2+ over Na+ compared with the human homologue Cav3.1, yet it exhibits a similar potent block of inward Na+ current by low external Ca2+ concentrations (i.e., the Ca2+ block effect). A comparison of the permeability features of TCav3 with other cloned channels suggests that Ca2+ block is a locus of evolutionary change in T-type channel cation permeation properties and that mammalian channels distinguish themselves from invertebrate ones by bearing both stronger Ca2+ block and higher Ca2+ selectivity. TCav3 is the most divergent metazoan T-type calcium channel and thus provides an evolutionary perspective on Cav3 channel structure-function properties, ion selectivity, and cellular physiology.

Calcium/calmodulin-dependent serine protein kinase CASK modulates the L-type calcium current

AimThe L-type voltage-gated calcium channel Cav1.2 mediates the calcium influx into cells upon membrane depolarization. The list of cardiopathies associated to Cav1.2 dysfunctions highlights the importance of this channel in cardiac physiology. Calcium/calmodulin-dependent serine protein kinase (CASK), expressed in cardiac cells, has been identified as a regulator of Cav2.2 channels in neurons, but no experiments have been performed to investigate its role in Cav1.2 regulation. Methods and resultsFull length or the distal C-terminal truncated of the pore-forming Cav1.2 channel (Cav1.2α1c), both present in cardiac cells, were expressed in TsA-201 cells. In addition, a shRNA silencer, or scramble as negative control, of CASK was co-transfected in order to silence CASK endogenously expressed. Three days post-transfection, the barium current was increased only for the truncated form without alteration of the steady state activation and inactivation biophysical properties. The calcium current, however, was increased after CASK silencing with both types of Cav1.2α1c subunits suggesting that, in absence of calcium, the distal C-terminal counteracts the CASK effect. Biochemistry experiments did not reveals neither an alteration of Cav1.2 channel protein expression after CASK silencing nor an interaction between Cav1.2α1c subunits and CASK. Nevertheless, after CASK silencing, single calcium channel recordings have shown an increase of the voltage-gated calcium channel Cav1.2 open probability explaining the increase of the whole-cell current. ConclusionThis study suggests CASK as a novel regulator of Cav1.2 via a modulation of the voltage-gated calcium channel Cav1.2 open probability.

Single calcium channel domain gating of synaptic vesicle fusion at fast synapses; analysis by graphic modeling

At fast-transmitting presynaptic terminals Ca2+ enter through voltage gated calcium channels (CaVs) and bind to a synaptic vesicle (SV) -associated calcium sensor (SV-sensor) to gate fusion and discharge. An open CaV generates a high-concentration plume, or nanodomain of Ca2+ that dissipates precipitously with distance from the pore. At most fast synapses, such as the frog neuromuscular junction (NMJ), the SV sensors are located sufficiently close to individual CaVs to be gated by single nanodomains. However, at others, such as the mature rodent calyx of Held (calyx of Held), the physiology is more complex with evidence that CaVs that are both close and distant from the SV sensor and it is argued that release is gated primarily by the overlapping Ca2+ nanodomains from many CaVs. We devised a 'graphic modeling' method to sum Ca2+ from individual CaVs located at varying distances from the SV-sensor to determine the SV release probability and also the fraction of that probability that can be attributed to single domain gating. This method was applied first to simplified, low and high CaV density model release sites and then to published data on the contrasting frog NMJ and the rodent calyx of Held native synapses. We report 3 main predictions: the SV-sensor is positioned very close to the point at which the SV fuses with the membrane; single domain-release gating predominates even at synapses where the SV abuts a large cluster of CaVs, and even relatively remote CaVs can contribute significantly to single domain-based gating.

Transmitter release is evoked with low probability predominately by calcium flux through single channel openings at the frog neuromuscular junction.

The quantitative relationship between presynaptic calcium influx and transmitter release critically depends on the spatial coupling of presynaptic calcium channels to synaptic vesicles. When there is a close association between calcium channels and synaptic vesicles, the flux through a single open calcium channel may be sufficient to trigger transmitter release. With increasing spatial distance, however, a larger number of open calcium channels might be required to contribute sufficient calcium ions to trigger vesicle fusion. Here we used a combination of pharmacological calcium channel block, high-resolution calcium imaging, postsynaptic recording, and 3D Monte Carlo reaction-diffusion simulations in the adult frog neuromuscular junction, to show that release of individual synaptic vesicles is predominately triggered by calcium ions entering the nerve terminal through the nearest open calcium channel. Furthermore, calcium ion flux through this channel has a low probability of triggering synaptic vesicle fusion (∼6%), even when multiple channels open in a single active zone. These mechanisms work to control the rare triggering of vesicle fusion in the frog neuromuscular junction from each of the tens of thousands of individual release sites at this large model synapse.

Synaptic vesicle tethering and the CaV2.2 distal C-terminal

Evidence that synaptic vesicles (SVs) can be gated by a single voltage sensitive calcium channel (CaV2.2) predict a molecular linking mechanism or “tether” (Stanley, 1993). Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel (Kaeser et al., 2011; Wong et al., 2013) while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site (Kaeser et al., 2011). Using a novel in vitro SV pull down binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137–2357; Wong et al., 2013). Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137–2299). To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349–2357) and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or “grabbed,” from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be “locked” close to the channel by a second attachment mechanism in preparation for single channel domain gating.

Size matters: formation and function of giant synapses

This symposium took place as a satellite of the Society for Neuroscience Meeting in New Orleans on Friday 12 October 2012, hosted by the Neuroscience Center of Excellence at Louisiana State University and sponsored by The Journal of Physiology and The Physiological Society. The meeting drew together several neuroscience communities with differing perspectives on development and function at large synapses in both mammalian and invertebrate nervous systems, including both CNS synapses and peripheral synapses of the neuromuscular junction (NMJ) and considering both pre- and postsynaptic interfaces. For a synapse, size is a clear predictor of its potential power in influencing the target (Petrof & Sherman, 2013). In most cases, single neurons integrate information from many thousands of synapses, but in some cases (such as the NMJ and the calyx of Held) a very high proportion of the synaptic input arises from one dominant axon terminal. Synaptic physiologists have favoured giant synapses for their accessibility, and much of our knowledge about synaptic transmission has been learned from these giant synapses, including the squid giant synapse and the frog NMJ. The unique accessibility of the NMJ has allowed developmental neurobiologists to elucidate the key factors required for synapse assembly. In recent times, a combination of technical breakthroughs in genetic labelling and imaging methods has illustrated that big is also beautiful, as evidenced by studies in which the formation of individual synapses can be followed in vivo. This series of symposium reviews focuses on the formation and then the function of large synapses across a variety of model systems. They include electrophysiological and imaging studies, with presentations about giant synapses from the NMJ to the retina, and from hippocampus to brainstem. The first review, from Tian & Wu (2013), considers the fly and worm model organisms to examine the molecular mechanisms by which ubiquitination regulates synaptogenesis and axonal responses after nerve injury. The ubiquitination cascade, in conjunction with autophagy, dynamically and specifically controls protein turnover to ensure normal synaptic development, clearance of injured distal axons and regeneration of proximal axons. Holcomb et al. (2013) have exploited the new scanning block-face electron microscopy to reconstruct both synaptic and postsynaptic targets of the calyx of Held synapse in order to ask fundamental questions about the origins of neuronal polarity and the ways in which this can guide the earliest stages of synapse formation. The developmental theme continues with a review of cerebellar climbing fibre synapse development, a well-established and elegant mode of synapse elimination in the mammalian CNS. This model is used here by Kano and colleagues (2013) to demonstrate how regulation of calcium transients is involved in the competitive strengthening or weakening of the synapse and implicated in later phases of elimination. From a functional perspective, synapses with differing synaptic strengths within the same network can perform differing functions. Petrof & Sherman (2013) explored the unique features of two classes of thalamocortical synapses with distinct integrative roles; large glutamatergic synapses predominate in transmission of information, while small glutamatergic synapses serve to modulate cortical function. The series concludes with reviews on transmitter release from some of the most intensely studied synaptic terminals. The strength and reliability of transmission at the NMJ is presented from both amphibian and mammalian viewpoints by Meriney & Dittrich (2013). Then the highly specialized sensory synapses of the ear and eye, which are adapted for high levels of exocytosis, are considered from the perspective of their respective neural codes. Kim et al. (2013) suggest that these data support the notion that single calcium channels can trigger exocytosis of multiple vesicles. The theme of contrasting form and function within synaptic structures is continued by Delvendahl et al. (2013) with the mossy fibre terminals of the hippocampus and cerebellum, where the low release probability of hippocampal mossy fibres contrasts with the high release probability of cerebellar mossy fibres. Finally, Neher & Taschenberger (2013) review the recent literature on intracellular calcium binding at the calyx of Held and present some unresolved questions about the mechanisms regulating the time course of the calcium transient.

Synaptic Ribbons Influence the Size and Frequency of Miniature-like Evoked Postsynaptic Currents

Nonspiking cells of several sensory systems respond to stimuli with graded changes in neurotransmitter release and possess specialized synaptic ribbons. Here, we show that manipulations to synaptic ribbons caused dramatic effects on mEPSC-like (mlEPSC) amplitude and frequency. Damage to rod-bipolar cell ribbons using fluorophore-assisted light inactivation resulted in the immediate reduction of mlEPSC amplitude and frequency, whereas the first evoked response after damage remained largely intact. The reduction in amplitude could not be recovered by increasing release frequency after ribbon damage. In parallel experiments, we looked at mlEPSCs from cones of hibernating ground squirrels, which exhibit dramatically smaller ribbons than awake animals. Fewer and smaller mlEPSCs were observed postsynaptic to cones from hibernating animals, although depolarized cones were able to generate larger mlEPSCs. Our results indicate that ribbon size may influence mlEPSC frequency and support a role for ribbons in coordinating multivesicular release.

Single-Channel Mechanism of Modulation of Calcium-Dependent Inactivation of the Voltage-Gated Calcium Channel Cav1.3 by its C-Terminus

Background: CaV1.3 is a L-type calcium channel pore subunit showing calcium-dependent inactivation (CDI). The full-length CaV1.3 isoform CaV1.342 contains a C-terminal modulator (CTM) domain that attenuates calcium-dependent inactivation at the whole-cell current level. CaV1.3 splice variants lack the CTM domain completely (CaV1.342A) or partially (CaV1.343S).Aim of the study: To analyse the role of the CTM domain for single-channel gating of CaV1.342, CaV1.342A and CaV1.343S in detail.Methods: We recorded single-channel calcium and barium currents carried by either CaV1.342, CaV1.342A and CaV1.343S plus auxiliary subunits expressed in HEK293 cells. Single-channel CDI was analysed according to Josephson et al. (J Physiol 2010;588:213). By Markov modelling transition rates between channel states were obtained.Results: As expected single-channel barium currents showed little inactivation during 150ms test pulses. Single-channel activity with barium was different with CaV1.342A being most and CaV1.342 least active (Bock et al., J Biol Chem 2011; 286:42736). This difference is due to CTM-dependent changes in the closed-to-open and open-to-closed transition rates across all test potentials examined (−20 to 0 mV). With calcium as charge carrier the CaV1.3 variants CaV1.342A and CaV1.343S lacking the CTM domain allowed for more pronounced CDI compared to full-length CaV1.342. Plotting the ratio of mean open times during late (MOT2) and early (MOT1) period of a depolarizing pulse against cumulative amount of calcium ions passing the channel reveals that a shortening of mean open times underlies CDI of CaV1.342A and CaV1.343S. The efficacy of calcium ions to induce this readout parameter of CDI was higher with the isoforms (partly) lacking CTM.Conclusion: CTM reduces the sensitivity of single CaV1.3 to CDI. Splice variants affecting the structure of CTM thus will influence the physiological function of e.g. cardiac pacemaker cells or neurons.

Prognosis of patients with coronary vasospasm after successful resuscitation from ventricular fibrillation

Ventricular fibrillation (VF) is an important cause of sudden cardiac death in patients with coronary vasospasm. Intensive medical treatment against coronary vasospasm is most important for prevention of VF recurrence, and the efficacy of implantable cardioverter-defibrillator (ICD) devices is controversial. We examined 19 consecutive patients with coronary spasm who had been successfully resuscitated from VF between 1993 and 2010, 13 of whom underwent ICD implantation. There were no apparent differences in the results of coronary angiography and acetylcholine provocation test or in medical therapy among them. Six patients underwent electrophysiological tests during the acetylcholine provocation test, and 3 of them showed ventricular arrhythmia. Three patients had recurrent VF under treatment with single calcium channel blocker (CCB), nitrates, and/or nicorandil. Two of them were without ICDs and were left in vegetative states, and the third was successfully resuscitated by an appropriate ICD discharge. The results of our study suggest that dual or multiple CCB treatment is preferable to prevent VF recurrence, and that combined treatment with ICD may be effective for some patients with coronary vasospasm who have been successfully resuscitated from VF. However, reasonable risk stratification for ICD implantation in these patients needs to be established in the future.

A global synergy of ions and voltage?

Membrane voltage and intracellular ion concentrations change under various cell states. Changes of both membrane potential and ion concentration can be sensed by the same elements in cell membranes. The calcium-activated potassium channels are a typical example. In hippocampal neurons, action potential waveforms are adapted to repetitive electrical inputs based on calcium-dependent repolarization. This may occur quickly or slowly. In the fast process, the duration of a single action potential becomes shorter. In the slow process, spike frequency and patterns are changed. The fast process is mainly achieved by activation of a class of Kv channel, a BK-type calcium-activated potassium channel. Its activities are regulated by both increase of intracellular calcium and depolarization. In fact, the BK channel has a voltage sensor domain and a cytoplasmic calcium-sensing domain. BK channels are often co-localized with voltage-gated calcium channels and can sense local and rapid calcium increase following opening of a single voltage-gated calcium channel during an action potential. The slower process of modification of neuronal firing depends on another class of potassium channel, the small conductance calcium-activated potassium channel (the SK channel), which binds calmodulin on the intracellular side. In contrast with the BK channel, the SK channel lacks intrinsic voltage dependence. Synergy between membrane voltage change and ions is not restricted to neurons and myocytes. In phagocytes such as the neutrophil or macrophage, reactive oxygen species are produced by the actions of NADPH oxidase. This activity induces remarkable depolarization since superoxide anions are transferred to the external space leaving protons inside. Thus both depolarization and intracellular acidification occur by the oxidase's activity. The voltage-gated proton (Hv) channel serves as an ideal player for cancelling both outcomes of the oxidase activity. The Hv channel is activated by both depolarization and intracellular acidification. Voltage dependence of steady-state open probability measured as the conductance–voltage (G–V) curve is shifted dependent on both intracellular and extracellular pH values. Recently, the molecular correlate of the Hv channel was identified. Knockout mice for the Hv channel exhibit reduced production of superoxide anions in neutrophils, consistent with the view that the Hv channel plays a role in promoting NADPH oxidase activity. More roles of the Hv channel in other cell types are also emerging. A study of knockout mice for the Hv channel led to the identification of unexpected roles in the signals of antibody production in B-lymphocytes. Another surprise was its presence and potential role in human sperm. Many ion channel species are expressed in sperm, but how ion channels contribute to sperm physiology, such as motility, has remained unknown. Patch clamping of sperm has now brought about a revolution in this field. This series of reviews covers three ion channel topics that explore the sites of synergy between voltage and ions. Jianmin Cui reviews recent findings of the structure–function relationship of the BK-type calcium-activated potassium channel (Cui, 2010). The interaction between S4, a key transmembrane segment of the voltage sensor domain, and the cytoplasmic metal ion-binding sites is one focus and the recently resolved X-ray structure of the cytoplasmic domain for calcium sensing is also discussed. Demaurex & Chemaly (2010) review the historical background of the Hv channel in leukocytes and recent molecular studies. Finally, Lishko & Kirichok (2010) review the recent discovery of the Hv channel in human sperm and its functional coupling with the CatSper channel, another class of voltage sensor containing channel that operates as an alkalization-activated calcium channel.

Loss of Intracellular and Intercellular Synchrony of Calcium Release in Systolic Heart Failure

Normal contraction of cardiac muscle requires a coordinated cellular mechanical response to the electric pacemaker signal that begins in the sinoatrial node and spreads through the conduction system and ultimately across the ventricular myocardium via gap junctions. In ventricular myocytes, depolarization by the action potential causes clusters of L-type calcium channels (LCCs) to open, allowing a small amount of Ca to enter the diadic cleft space between the sarcolemma and the sarcoplasmic reticulum (SR). When the Ca concentration in this space becomes sufficiently high to gate ryanodine receptors (RyRs), they open and release SR Ca into the cytoplasm, so that it can bind to and activate myofilaments, resulting in force development. Each cluster of LCCs on the sarcolemma, its opposing cluster of RyRs across the 10- to 12-nm diameter diadic cleft space, and associated SR regulatory proteins is a couplon.1 According to the local control theory of cardiac excitation-contraction (EC) coupling, couplons are activated independently. Independent activation of couplons depends on the extent of LCC activation, providing a mechanism for regulation of contractile force by stochastic recruitment of couplons.2 In systolic heart failure, numerous cellular defects in EC coupling and Ca regulation have been identified that contribute to contractile dysfunction (and ventricular arrhythmias), although there is a host of other major abnormalities leading to loss of force development, including fibrosis,3 defective energy metabolism,4 pH changes,5 heterogeneous conductance across connexin hemichannels,6 and defects in sarcomeric proteins.7 Thus, it is important to keep in mind that no single defect can explain the contractile dysfunction of systolic failure. Article see p 223 Recent studies of defective EC coupling in heart failure have addressed not only reductions in the extent and kinetics of Ca released in response to the action potential (ie, reduced recruitment of couplons and …

Localization of Calcium Channels at the Calyx of Held

The Calyx of Held is a giant synapse in the auditory pathway. Due to its large size and its geometry it makes an excellent model system for studying the biophysical properties of synaptic transmission. Previous studies have demonstrated that the release rate of synaptic vesicles is governed by the distance between single synaptic vesicles and calcium channels. Therefore knowing the localization of presynaptic calcium channels is essential to understand the coupling of synaptic vesicle release with calcium influx into the calyx. Prior studies have attempted to characterize calcium channel distribution in the Calyx, however results have been inconclusive in providing an accurate picture. In order to conclusively localize calcium channels we are exploring several possibilities to stain calcium channels for fluorescence and electron microscopy studies. The first approach is to express genetically tagged P/Q-type calcium channels in the Calyx utilizing an adenovirus expression system. We have tried an eGFP-tagged P/Q-type calcium channel and found that the eGFP accumulates in the nuclei of the cells indicating cleavage of the tags. The tag also reduced calcium currents twofold compared to untagged channel. Our second approach was to screen several constructs with HA epitope tags inserted into different positions on the P/Q-type calcium channel. We found that extracellular tags frequently impair channel function. However we did obtain constructs which showed current upon expression in HEK cells. Unfortunately accessibility of the tag for the antibody is impaired in neurons. Intracellular tags are accesible in primary neurons and behave similar to untagged channel electrophysiologically, but the staining is difficult to interpret because tagged overexpressed channels accumulate in intracellular compartments. In parallel experiments we have screened several antibodies against P/Q-type calcium channels aiming to find one that gives clean staining for high-resolution microscopy. Funding: LSHM-CT-1005-019055.

Nanodomain Coupling between Ca2+ Channels and Ca2+ Sensors Promotes Fast and Efficient Transmitter Release at a Cortical GABAergic Synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.

Mechanism of Cav1.2 channel modulation by the amino terminus of cardiac β2‐subunits

L-type calcium channels are composed of a pore, alpha1c (Ca(V)1.2), and accessory beta- and alpha2delta-subunits. The beta-subunit core structure was recently resolved at high resolution, providing important information on many functional aspects of channel modulation. In this study we reveal differential novel effects of five beta2-subunits isoforms expressed in human heart (beta(2a-e)) on the single L-type calcium channel current. These splice variants differ only by amino-terminal length and amino acid composition. Single-channel modulation by beta2-subunit isoforms was investigated in HEK293 cells expressing the recombinant L-type ion conducting pore. All beta2-subunits increased open probability, availability, and peak current with a highly consistent rank order (beta2a approximately = beta2b > beta2e approximately = beta2c > beta2d). We show graded modulation of some transition rates within and between deep-closed and inactivated states. The extent of modulation correlates strongly with the length of amino-terminal domains. Two mutant beta2-subunits that imitate the natural span related to length confirm this conclusion. The data show that the length of amino termini is a relevant physiological mechanism for channel closure and inactivation, and that natural alternative splicing exploits this principle for modulation of the gating properties of calcium channels.

Imaging single-channel calcium microdomains

The Ca 2+ microdomains generated around the mouth of open ion channels represent the basic building blocks from which cytosolic Ca 2+ signals are constructed. Recent improvements in optical imaging techniques now allow these microdomains to be visualized as single channel calcium fluorescence transients (SCCaFTs), providing information about channel properties that was previously accessible only by electrophysiological patch-clamp recordings. We review recent advances in single channel Ca 2+ imaging methodologies, with emphasis on total internal reflection fluorescence microscopy (TIRFM) as the technique of choice for recording SCCaFTs from voltage- and ligand-gated plasmalemmal ion channels. This technique of ‘optical patch-clamp recording’ is massively parallel, permitting simultaneous imaging of hundreds of channels; provides millisecond resolution of gating kinetics together with sub-micron spatial resolution of channel locations; and is applicable to diverse families of membrane channels that display partial permeability to Ca 2+ ions.

Long-Term and Immediate Effect of Testosterone on Single T-Type Calcium Channel in Neonatal Rat Cardiomyocytes

In the cardiovascular system, T-type calcium channels play an important role for the intracellular calcium homeostasis and spontaneous pacemaker activity and are involved in the progression of structural heart diseases. Androgens influence the cardiovascular physiology and pathophysiology. However, their effect on native T-type calcium currents (I(Ca,T)) remains unclear. To test the chronic effect of testosterone on the cardiac I(Ca,T), cultured neonatal rat ventricular cardiomyocytes were treated with testosterone (1 nM-10 microM) for 24-30 h. Current measurements were performed after testosterone washout to exclude any acute testosterone effects. Testosterone (100 nm) pretreatment significantly increased whole-cell I(Ca,T) density from 1.26 +/- 0.48 pA/pF (n = 8) to 5.06 +/- 1.75 pA/pF (n = 7; P < 0.05) and accelerated beating rate. This was attributed to both increased expression levels of the pore-forming subunits Ca(v)3.1 and Ca(v)3.2 and increased T-type single-channel activity. On single-channel level, the increase of the ensemble average current by testosterone vs. time-matched controls was due to an increased availability (58.1 +/- 4.2 vs. 21.5 +/- 4.0%, P < 0.01) and open probability (2.78 +/- 0.29 vs. 0.85 +/- 0.23%, P < 0.01). Cotreatment with the selective testosterone receptor antagonist flutamide (10 mum) prevented these chronic testosterone-induced effects. Conversely, acute application of testosterone (10 microM) decreased T-type single-channel activity in testosterone pretreated cells by reducing the open probability (0.78 +/- 0.13 vs. 2.91 +/- 0.38%, P < 0.01), availability (23.6 +/- 3.3 vs. 57.6 +/- 4.5%, P < 0.01), and peak current (-20 +/- 4 vs. -58 +/- 4 fA, P < 0.01). Flutamide (10 microM) did not abolish the testosterone-induced acute block of T-type calcium channels. Our results indicate that long-term testosterone treatment increases, whereas acute testosterone decreases neonatal rat T-type calcium currents. These effects seem to be mediated by a genomic chronic stimulation and a nongenomic acute inhibitory action.