Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte

G-protein-coupled receptor signalling has been suggested to be voltage dependent in a number of cell types; however, the limits of sensitivity of this potentially important phenomenon are unknown. Using the non-excitable rat megakaryocyte as a model system, we now show that P2Y receptor-evoked Ca²⁺ mobilization is controlled by membrane voltage in a graded and bipolar manner without evidence for a discrete threshold potential. Throughout the range of potentials studied, the peak increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to depolarization was always larger than the maximal reduction in [Ca²⁺]_i following an equivalent amplitude hyperpolarization. Significant [Ca²⁺]_i increases were observed in response to small amplitude (<5 mV, 5 s duration) or short duration (25 ms, 135 mV) depolarizations. Individual cardiac action potential waveforms were also able to repeatedly potentiate P2Y receptor-evoked Ca²⁺ release and the response to trains of normally paced stimuli fused to generate prolonged [Ca²⁺]_i increases. Furthermore, elevation of the temperature to physiological levels (36°C) resulted in a more sustained depolarization-evoked Ca²⁺ increase compared with more transient or oscillatory responses at 20–24°C. The ability of signalling via a G-protein-coupled receptor to be potentiated by action potential waveforms and small amplitude depolarizations has broad implications in excitable and non-excitable tissues.     

Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells

Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca_V1.3 Ca²⁺ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca²⁺ channels in immature mouse IHCs under near-physiological recording conditions. Ca_V1.3 Ca²⁺ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca²⁺ action potential activity characteristic of these immature cells. The Ca_V1.3 Ca²⁺ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca²⁺ currents indicated that very few Ca²⁺ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca²⁺ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca²⁺ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.     

Ca2+-NFATc1 signaling is an essential axis of osteoclast differentiation

Summary:  Osteoclasts are unique, multinucleated giant cells that decalcify and degrade the bone matrix. They originate from hematopoietic cells and their differentiation is dependent on a tumor necrosis factor (TNF) family cytokine, receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL), as well as macrophage-colony stimulating factor (M-CSF). Recent studies have unveiled the precise molecular mechanism underlying osteoclastogenesis. In particular, the discovery of nuclear factor of activated T cells c1 (NFATc1), the master regulator of osteoclastogenesis, has proven to be a breakthrough in this field. NFATc1 is activated by Ca²⁺ signaling induced by the activation of the immunoglobulin-like receptor signaling associated with immunoreceptor tyrosine-based activation motif (ITAM)-harboring adapters. The long-lasting Ca²⁺ oscillation, which is evident during osteoclastogenesis, may ensure the robust induction of NFATc1 through an autoamplification mechanism. Thus, intracellular Ca²⁺ is a critical attribute of osteoclastogenic signaling. In addition, osteoclasts are exposed to a very high extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the bone microenvironment and respond to the change in [Ca²⁺]_o by increasing the intracellular Ca²⁺, which regulates diverse cellular functions. Investigation of the molecular mechanisms underlying the regulation of intracellular Ca²⁺ dynamics may open up new directions for therapeutic strategies in bone disease.     

Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function

The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca²⁺ currents and Ca²⁺-activated K⁺ channels (K_Ca channels) is important for Purkinje cell activity, but how many different K_Ca channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca²⁺ channel and K_Ca channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca²⁺ entering through P-type voltage-gated Ca²⁺ channels activates both small-conductance (SK) and large-conductance (BK) K_Ca channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response.     

'Delayed' endocytosis is regulated by extracellular Ca2+ in snake motor boutons

When cooled below ≈7 °C, recently endocytosed vesicles in the motor terminals of the garter snake fail to shed their clathrin coats. Perhaps as a result, the terminals complete only about one-half of the compensatory endocytosis expected after a given period of stimulation. Upon return to room temperature (RT), endocytosis resumes immediately and is complete within minutes. This 'delayed' endocytosis following release from cold block provides an opportunity to study clathrin-dependent endocytotic mechanisms in temporal isolation from those events, such as Ca²⁺ entry and consequent exocytosis, that are normally associated with the activation of nerve terminals. We have taken advantage of clathrin decoating blockade to examine the rate, temperature dependence and extracellular Ca²⁺ dependence of endocytosis at the snake nerve-muscle synapse. Endocytosis was fast at RT (complete in < 1 min) and markedly faster still at 35 °C. Moreover, the rate of endocytosis varied significantly with change in [Ca²⁺]_o; the rate at 7.2 mM (single exponential time constant, ≈3 s) was approximately double that at 0 mM (single exponential time constant, ≈7 s). Thus, membrane retrieval via clathrin is rapid and, due to its dependence on [Ca²⁺]_o, potentially regulated by changes in the milieu of the synaptic cleft during neural activity.     

Voltage-dependent inactivation of l-type ca2+ Currents in Guinea-Pig Ventricular Myocytes

The objective of this study was to describe the kinetics of voltage-dependent inactivation of native cardiac L-type Ca²⁺ currents. Whole-cell currents were recorded from guinea-pig isolated ventricular myocytes. Voltage-dependent inactivation was separated from Ca²⁺-dependent inactivation by replacing extracellular Ca²⁺ with Mg²⁺ and recording outward currents through Ca²⁺ channels. Voltage-dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage-dependent inactivation occurred with τ_f≈30 ms and τ_s≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca²⁺ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time-independent component of the Ca²⁺ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca²⁺ channel current availability at positive membrane potentials and thus a U-shaped availability-voltage ( A-V) relationship. It is concluded that β-adrenergic stimulation altered the reaction of native cardiac L-type Ca²⁺ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.     

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca²⁺ sparks stimulate nearby Ca²⁺-activated K⁺ (BK) channels that hyperpolarize the membrane and close L-type Ca²⁺ channels. We tested the contribution of L-type Ca_v1.2 channels to Ca²⁺ spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca_v1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca_v1.2 channel gene inactivation reduced Ca²⁺ spark frequency and amplitude by ∼50% and ∼80%, respectively. These effects were associated with lower global cytosolic Ca²⁺ levels and reduced sarcoplasmic reticulum (SR) Ca²⁺ load. Elevating cytosolic Ca²⁺ levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca_v1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca²⁺ sparks. Instead, Ca_v1.2 channels contribute to global cytosolic [Ca²⁺], which in turn influences luminal SR calcium and thus Ca²⁺ sparks.     

Physiological modulation of inactivation in L-type Ca2+ channels: one switch

The relative contributions of voltage- and Ca²⁺-dependent mechanisms of inactivation to the decay of L-type Ca²⁺ channel currents ( I _CaL) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage-dependent inactivation (VDI) was thought to be slow and Ca²⁺-dependent inactivation (CDI) resulting from Ca²⁺ influx and Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca²⁺ entry and the contribution of I _CaL to the cardiac action potential. Physiological modulation of I _CaL by β-adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca²⁺ channel activity, Ca²⁺ influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of I _CaL. Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by β-adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of I _CaL are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of I _CaL to the action potential will be determined largely by voltage and by Ca²⁺ following β-adrenergic stimulation.     

Role of extracellular Ca2+ in gating of CaV1.2 channels

We examined changes in ionic and gating currents in Ca_V1.2 channels when extracellular Ca²⁺ was reduced from 10 m m to 0.1 μ m . Saturating gating currents decreased by two-thirds ( K _D≈ 40 μ m ) and ionic currents increased 5-fold ( K _D≈ 0.5 μ m ) due to increasing Na⁺ conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca²⁺], which appeared to reflect the rapid activation of channels that were not blocked by Ca²⁺ and a slower reversal of Ca²⁺ blockade of the remaining channels. Removal of Ca²⁺ following inactivation of Ca²⁺ currents showed that Na⁺ currents were not affected by Ca²⁺-dependent inactivation. Ca²⁺-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca²⁺ conductance and Ca²⁺-dependent inactivation depend on extracellular Ca²⁺ and are linked to changes in selectivity.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

Buffer Kinetics Shape the Spatiotemporal Patterns of IP3-Evoked Ca2+ Signals

Ca²⁺ liberation through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca²⁺ signals. IP₃Rs display Ca²⁺-induced Ca²⁺ release (CICR), but are grouped in clusters so that regenerative Ca²⁺ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca²⁺ diffusion and CICR. We used confocal microscopy and photoreleased IP₃ in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca²⁺ buffers. EGTA (a buffer with slow 'on-rate') speeded Ca²⁺ signals and 'balkanized' Ca²⁺ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca²⁺ responses and promoted 'globalization' of spatially uniform Ca²⁺ signals. These actions are likely to arise through differential effects on Ca²⁺ feedback within and between IP₃R clusters, because Ca²⁺ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca²⁺-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP₃-evoked Ca²⁺ signals for specific physiological roles.     

Small conductance Ca2+-activated K+ channels and calmodulin

Small conductance Ca²⁺-activated K⁺ channels (SK channels) contribute to the long lasting afterhyperpolarization (AHP) that follows an action potential in many central neurones. The biophysical and pharmacological attributes of cloned SK channels strongly suggest that one or more of them underlie the medium component of the AHP that regulates interspike interval and plays an important role in setting tonic firing frequency. The cloned SK channels comprise a distinct subfamily of K⁺ channels. Heterologously expressed SK channels recapitulate the biophysical and pharmacological hallmarks of native SK channels, being gated solely by intracellular Ca²⁺ ions with no voltage dependence to their gating, small unitary conductance values and sensitivity to the bee venom peptide toxin, apamin. Molecular, biochemical and electrophysiological studies have revealed that Ca²⁺ gating in SK channels is due to heteromeric assembly of the SK α pore-forming subunits with calmodulin (CaM). Ca²⁺ binding to the N-terminal E–F hands of CaM is responsible for SK channel gating. Crystallographic studies suggest that SK channels gate as a dimer-of-dimers, and that the physical gate of SK channels resides at or near the selectivity filter of the channels. In addition, Ca²⁺-independent interactions between the SK channel α subunits and CaM are necessary for proper membrane trafficking.     

Role of aspartate 298 in mouse 5-HT3A receptor gating and modulation by extracellular Ca2+

The TM2–TM3 extracellular loop is critical for activation of the Cys-loop family of ligand-gated ion channels. The contribution of aspartate 298 (D298), an amino acid that links the transmembrane domain 2 (TM2) to the TM2–TM3 loop, in mouse 5-hydroxytryptamine_3A (5-HT_3A) receptor function was probed with site-directed mutagenesis in the present study. This negatively charged residue was replaced with an alanine to neutralize the charge, with a glutamate to conserve the charge, or with an arginine to reverse the charge. Human embryonic kidney 293 (HEK 293) cells transfected with the wild-type and mutant receptors were studied by combining whole-cell patch-clamp recording with fast agonist application. The D→A or D→R mutations resulted in a receptor with reduced 5-HT potency, and accelerated kinetics of desensitization and deactivation. In addition, the efficacy of partial agonists was reduced by the D→A mutation. The D→E mutation produced a receptor with properties similar to those of the wild-type receptor. In addition, the potential role of this residue in modulation of the receptor by extracellular calcium ([Ca²⁺]_o) was investigated. Increasing [Ca²⁺]_o inhibited 5-HT-activated currents and altered receptor kinetics in a similar manner in the wild-type and D298E receptors, and this alteration was eliminated by the D→A and D→R mutations. Our data suggest that the charge at D298 participates in transitions between functional states of the 5-HT_3A receptor, and provide evidence that the charge of the side-chain at residue D298 contributes to channel gating kinetics and is crucial for Ca²⁺ modulation.     

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca²⁺ stores, following the release of Ca²⁺ during intracellular signalling, triggers the Ca²⁺ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca²⁺]_i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca²⁺ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca²⁺ entry in the form of motes.     

Spino-dendritic cross-talk in rodent Purkinje neurons mediated by endogenous Ca2+-binding proteins

The range of actions of the second messenger Ca²⁺ is a key determinant of neuronal excitability and plasticity. For dendritic spines, there is on-going debate regarding how diffusional efflux of Ca²⁺ affects spine signalling. However, the consequences of spino-dendritic coupling for dendritic Ca²⁺ homeostasis and downstream signalling cascades have not been explored to date. We addressed this question by four-dimensional computer simulations, which were based on Ca²⁺-imaging data from mice that either express or lack distinct endogenous Ca²⁺-binding proteins. Our simulations revealed that single active spines do not affect dendritic Ca²⁺ signalling. Neighbouring, coactive spines, however, induce sizeable increases in dendritic [Ca²⁺]_i when they process slow synaptic Ca²⁺ signals, such as those implicated in the induction of long-term plasticity. This spino-dendritic coupling is mediated by buffered diffusion, specifically by diffusing calbindin-bound Ca²⁺. This represents a central mechanism for activating calmodulin in dendritic shafts and therefore a novel form of signal integration in spiny dendrites.     

The effects of intracellular Ca2+ on cardiac K+ channel expression and activity: novel insights from genetically altered mice

We tested the hypothesis that chronic changes in intracellular Ca²⁺ (Ca²⁺_i) can result in changes in ion channel expression; this represents a novel mechanism of crosstalk between changes in Ca²⁺ cycling proteins and the cardiac action potential (AP) profile. We used a transgenic mouse with cardiac-specific overexpression of sarcoplasmic reticulum Ca²⁺ ATPase (SERCA) isoform 1a (SERCA1a OE) with a significant alteration of SERCA protein levels without cardiac hypertrophy or failure. Here, we report significant changes in the expression of a transient outward K⁺ current ( I _to,f), a slowly inactivating K⁺ current ( I _K,slow) and the steady state current ( I _SS) in the transgenic mice with resultant prolongation in cardiac action potential duration (APD) compared with the wild-type littermates. In addition, there was a significant prolongation of the QT interval on surface electrocardiograms in SERCA1a OE mice. The electrophysiological changes, which correlated with changes in Ca²⁺_i, were further corroborated by measuring the levels of ion channel protein expression. To recapitulate the in vivo experiments, the effects of changes in Ca²⁺_i on ion channel expression were further tested in cultured adult and neonatal mouse cardiac myocytes. We conclude that a primary defect in Ca²⁺ handling proteins without cardiac hypertrophy or failure may produce profound changes in K⁺ channel expression and activity as well as cardiac AP.     

Descending vasa recta pericytes express voltage operated Na+ conductance in the rat

We studied the properties of a voltage-operated Na⁺ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole-cell patch-clamp recordings revealed a depolarization-induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na⁺ but not Ca⁺ from the extracellular buffer. The Na⁺ current ( I _Na) is highly sensitive to tetrodotoxin  (TTX, K _d= 2.2 n m )  . At high concentrations, mibefradil (10 μ m ) and Ni⁺ (1 m m ) blocked I _Na. I _Na was insensitive to nifedipine (10 μ m ). The L-type Ca⁺ channel activator FPL-64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half-maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na⁺ channel activator, veratridine (100 μ m ), reduced peak inward I _Na and prevented inactivation. We conclude that a TTX-sensitive voltage-operated Na⁺ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.     

T-type Ca2+ channels encode prior neuronal activity as modulated recovery rates

T-type Ca²⁺ channels give rise to low-threshold inward currents that are central determinants of neuronal excitability. The availability of T-type Ca²⁺ channels is strongly influenced by voltage-dependent inactivation and recovery from inactivation. Here, we show that native and cloned T-type Ca²⁺ channel subunits selectively encode specific aspects of prior membrane potential changes via a powerful modulation of the rates with which these channels recover from inactivation. Increasing the duration of subthreshold (−70 to −55 mV) conditioning depolarizations caused a pronounced slowing of subsequent recovery from inactivation of both cloned (Ca_v3.1–3.3) and native T-type channels (thalamic neurones). The scaling of recovery rates with increasing duration of conditioning depolarizations could be well described by a power law function. Different T-type channel isoforms exhibited overlapping but complementary ranges of recovery rates. Intriguingly, scaling of recovery rates was dramatically reduced in Ca_v3.2 and Ca_v3.3, but not Ca_v3.1 subunits, when mock action potentials were superimposed on conditioning depolarizations. Our results suggest that different T-type channel subunits exhibit dramatic differences in scaling relationships, in addition to well-described differences in other biophysical properties. Furthermore, the availability of T-type channels is powerfully modulated over time, depending on the patterns of prior activity that these channels have encountered. These data provide a novel mechanism for cellular short-term plasticity on the millisecond to second time scale that relies on biophysical properties of specific T-type Ca²⁺ channel subunits.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

Increased Ca2+ influx through Na+/Ca2+ exchanger during long-term facilitation at crayfish neuromuscular junctions

Intense motor neuron activity induces a long-term facilitation (LTF) of synaptic transmission at crayfish neuromuscular junctions (NMJs) that is accompanied by an increase in the accumulation of presynaptic Ca²⁺ ions during a test train of action potentials. It is natural to assume that the increased Ca²⁺ influx during action potentials is directly responsible for the increased transmitter release in LTF, especially as the magnitudes of LTF and increased Ca²⁺ influx are positively correlated. However, our results indicate that the elevated Ca²⁺ entry occurs through the reverse mode operation of presynaptic Na⁺/Ca²⁺ exchangers that are activated by an LTF-inducing tetanus. Inhibition of Na⁺/Ca²⁺ exchange blocks this additional Ca²⁺ influx without affecting LTF, showing that LTF is not a consequence of the regulation of these transporters and is not directly related to the increase in [Ca²⁺]_i reached during a train of action potentials. Their correlation is probably due to both being induced independently by the strong [Ca²⁺]_i elevation accompanying LTF-inducing stimuli. Our results reveal a new form of regulation of neuronal Na⁺/Ca²⁺ exchange that does not directly alter the strength of synaptic transmission.