Voltage-dependent Facilitation Research Articles

Optically Probing the Mechanism of Voltage-Dependent Facilitation in CaV1.2 Channels

An important regulatory mechanism of excitation-evoked Ca2+ influx is voltage-dependent, or pre-pulse, facilitation (VDF): a memory property of some CaV channels, whereby channel activation is enhanced following a pre-conditioning pulse of sufficient duration and amplitude. While VDF can greatly increase Ca2+ influx and confer memory of excitation, the molecular determinants for its voltage- and time-dependent properties are unclear. We posited that VDF emerges from the activity of one, or more, of the four homologous, but non-identical, voltage-sensing domains (VSDs) of the pore-forming α1 subunit. Using voltage clamp fluorometry, we optically tracked the activation of each VSD in human CaV1.2 (α1C) channels expressed in Xenopus oocytes in association with β3 subunits, voltage-clamped by the cut-open oocyte method, in the absence or presence of facilitating pre-pulses (80 mV, 200 ms). Ba2+ was used as the charge carrier to exclude Ca2+-dependent processes. Pre-pulses increased the activation of VSD-I and -II at 20 mV by ∼60% and accelerated VSD-I activation kinetics by ∼10-fold. VSD-III activation was shifted to more hyperpolarized potentials by ∼20mV and doubled in effective valence. In non-facilitated α1C/β3 channels, VSD-II and -III are weakly-coupled to channel opening (Savalli et al., 2016 J Gen Physiol); pre-pulse-induced facilitation of their activation suggests that VDF engages these VSDs with the channel gate. This premise is supported by the finding that association of the α2δ-1 subunit (which mimics facilitation; Platano et al., 2000 Biophys J) strongly couples VSD-II and -III to the channel gate (Pantazis et al., 2014 PNAS). Since VSD-I (i) is also potentiated by pre-pulses, but (ii) is weakly-coupled to channel opening in both non-facilitated α1C/β3 and “constitutively facilitated” α1C/β3/α2δ channels, our work highlights it as a plausible candidate for the structural determinant of VDF.

Dopamine D1 receptor modulation of calcium channel currents in horizontal cells of mouse retina.

Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 μM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 μM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 μM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 μM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gβγ-blocking peptide suggested involvement of Gβγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gβγ proteins in horizontal cells.

Regulation of Cardiac L-Type Ca 2+ Channel Ca V 1.2 Via the β-Adrenergic-cAMP-Protein Kinase A Pathway

In the heart, adrenergic stimulation activates the β-adrenergic receptors coupled to the heterotrimeric stimulatory Gs protein, followed by subsequent activation of adenylyl cyclase, elevation of cyclic AMP levels, and protein kinase A (PKA) activation. One of the main targets for PKA modulation is the cardiac L-type Ca²⁺ channel (CaV1.2) located in the plasma membrane and along the T-tubules, which mediates Ca²⁺ entry into cardiomyocytes. β-Adrenergic receptor activation increases the Ca²⁺ current via CaV1.2 channels and is responsible for the positive ionotropic effect of adrenergic stimulation. Despite decades of research, the molecular mechanism underlying this modulation has not been fully resolved. On the contrary, initial reports of identification of key components in this modulation were later refuted using advanced model systems, especially transgenic animals. Some of the cardinal debated issues include details of specific subunits and residues in CaV1.2 phosphorylated by PKA, the nature, extent, and role of post-translational processing of CaV1.2, and the role of auxiliary proteins (such as A kinase anchoring proteins) involved in PKA regulation. In addition, the previously proposed crucial role of PKA in modulation of unstimulated Ca²⁺ current in the absence of β-adrenergic receptor stimulation and in voltage-dependent facilitation of CaV1.2 remains uncertain. Full reconstitution of the β-adrenergic receptor signaling pathway in heterologous expression systems remains an unmet challenge. This review summarizes the past and new findings, the mechanisms proposed and later proven, rejected or disputed, and emphasizes the essential issues that remain unresolved.

Temperature and functional plasticity of L-type Ca2+ channels in Drosophila

The question of optimization of ion channel function to surrounding temperatures in poikilothermic organisms remains largely uninvestigated. Here, we addressed it by studying the temperature-dependence of L-type Ca2+ channels (LTCCs) in Drosophila larval muscles in the context of their modulation by protein kinase A (PKA). LTCC currents were recorded between 4 and 30°C. Different aspects of LTCC function reached maxima between 15 and 25°C: conductance, tail current amplitude, inactivation rate, and the level of basal up-regulation by PKA (26% at 21°C). Anomalous temperature-dependencies of LTCC conductance and kinetics were similar in control and in the presence of the PKA inhibitor H-89. Analysis of deactivation kinetics revealed excessive tail currents at lower temperatures (up to 15°C), indicative of voltage-dependent facilitation of LTCCs. Tail current magnitude gradually decreased with temperature from a maximum at 15°C until a nearly complete disappearance at 30°C. Elimination of excessive tail currents at higher temperatures coincided with unusual slowing of inactivation, suggesting disruption of the facilitation by rising temperature, possibly through depletion of the pool of contributing channels. Overall, these results suggest the presence of a physiological plasticity optimum of LTCC function in the temperature range of normal Drosophila development.

Harmonin enhances voltage‐dependent facilitation of Cav1.3 channels and synchronous exocytosis in mouse inner hair cells

Cav1.3 channels mediate Ca(2+) influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain-containing protein that interacts with the C-terminus of the Cav1.3 α1 subunit (α11.3) and controls cell surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain-containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α11.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cav β1 than with the β2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the 'deaf-circler' dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca(2+) influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca(2+) channels and exocytosis.

Distinct localization and modulation of Cav1.2 and Cav1.3 L‐type Ca2+ channels in mouse sinoatrial node

Dysregulation of L-type Ca(2+) currents in sinoatrial nodal (SAN) cells causes cardiac arrhythmia. Both Ca(v)1.2 and Ca(v)1.3 channels mediate sinoatrial L-type currents. Whether these channels exhibit differences in modulation and localization, which could affect their contribution to pacemaking, is unknown. In this study, we characterized voltage-dependent facilitation (VDF) and subcellular localization of Ca(v)1.2 and Ca(v)1.3 channels in mouse SAN cells and determined how these properties of Ca(v)1.3 affect sinoatrial pacemaking in a mathematical model. Whole cell Ba(2+) currents were recorded from SAN cells from mice carrying a point mutation that renders Ca(v)1.2 channels relatively insensitive to dihydropyridine antagonists. The Ca(v)1.2-mediated current was isolated in the presence of nimodipine (1 μm), which was subtracted from the total current to yield the Ca(v)1.3 component. With strong depolarizations (+80 mV), Ca(v)1.2 underwent significantly stronger inactivation than Ca(v)1.3. VDF of Ca(v)1.3 was evident during recovery from inactivation at a time when Ca(v)1.2 remained inactivated. By immunofluorescence, Ca(v)1.3 colocalized with ryanodine receptors in sarcomeric structures while Ca(v)1.2 was largely restricted to the delimiting plasma membrane. Ca(v)1.3 VDF enhanced recovery of pacemaker activity after pauses and positively regulated pacemaking during slow heart rate in a numerical model of mouse SAN automaticity, including preferential coupling of Ca(v)1.3 to ryanodine receptor-mediated Ca(2+) release. We conclude that strong VDF and colocalization with ryanodine receptors in mouse SAN cells are unique properties that may underlie a specific role for Ca(v)1.3 in opposing abnormal slowing of heart rate.

Facilitation and Ca2+-dependent Inactivation Are Modified by Mutation of the Cav1.2 Channel IQ Motif

The heart muscle responds to physiological needs with a short-term modulation of cardiac contractility. This process is determined mainly by properties of the cardiac L-type Ca(2+) channel (Ca(v)1.2), including facilitation and Ca(2+)-dependent inactivation (CDI). Both facilitation and CDI involve the interaction of calmodulin with the IQ motif of the Ca(v)1.2 channel, especially with Ile-1624. To verify this hypothesis, we created a mouse line in which Ile-1624 was mutated to Glu (Ca(v)1.2(I1624E) mice). Homozygous Ca(v)1.2(I1624E) mice were not viable. Therefore, we inactivated the floxed Ca(v)1.2 gene of heterozygous Ca(v)1.2(I1624E) mice by the α-myosin heavy chain-MerCreMer system. The resulting I/E mice were studied at day 10 after treatment with tamoxifen. Electrophysiological recordings in ventricular cardiomyocytes revealed a reduced Ca(v)1.2 current (I(Ca)) density in I/E mice. Steady-state inactivation and recovery from inactivation were modified in I/E versus control mice. In addition, voltage-dependent facilitation was almost abolished in I/E mice. The time course of I(Ca) inactivation in I/E mice was not influenced by the use of Ba(2+) as a charge carrier. Using 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid as a chelating agent for intracellular Ca(2+), inactivation of I(Ca) was slowed down in control but not I/E mice. The results show that the I/E mutation abolishes Ca(2+)/calmodulin-dependent regulation of Ca(v)1.2. The Ca(v)1.2(I1624E) mutation transforms the channel to a phenotype mimicking CDI.

Splice-variant changes of the CaV3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (CaV3.1, CaV3.2,CaV3 .3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize CaV3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length CaV3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac CaV3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression ofCaV3 .2(-25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (-25) exon variants, (3) in the adult stage of hypertensive rats there is a both an increase in overallCaV3 .2 expression and a shift towards expression of CaV3.2(+25) containing channels as the predominant form, and (4) alternative splicing confers a variant-specific voltage-dependent facilitation ofCaV3 .2 channels. We conclude that CaV3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.

Facilitation of murine cardiac L-type Ca v 1.2 channel is modulated by Calmodulin kinase II–dependent phosphorylation of S1512 and S1570

Activity-dependent means of altering calcium (Ca(2)(+)) influx are assumed to be of great physiological consequence, although definitive tests of this assumption have only begun to emerge. Facilitation and inactivation offer two opposing, activity-dependent means of altering Ca(2+) influx via cardiac Ca(v)1.2 calcium channels. Voltage- and frequency-dependent facilitation of Ca(v)1.2 has been reported to depend on Calmodulin (CaM) and/or the activity of Calmodulin kinase II (CaMKII). Several sites within the cardiac L-type calcium channel complex have been proposed as the targets of CaMKII. Here, we generated mice with knockin mutations of alpha(1)1.2 S1512 and S1570 phosphorylation sites [sine facilitation (SF) mice]. Homocygote SF mice were viable and reproduced in a Mendelian ratio. Voltage-dependent facilitation in ventricular cardiomyocytes carrying the SF mutation was decreased from 1.58- to 1.18-fold. The CaMKII inhibitor KN-93 reduced facilitation to 1.28 in control cardiomyocytes. SF mutation negatively shifted the voltage-dependent inactivation and slowed recovery from inactivation, thereby making fewer channels available for activation. Telemetric ECG recordings at different heart rates showed that QT time decreased significantly more in SF than in control mice at higher rates. Our results strongly support the notion that CaMKII-dependent phosphorylation of Cav1.2 at S1512 and S1570 mediates Ca(2+) current facilitation in the murine heart.

Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called "voltage-dependent facilitation" observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory.

Effects of local anesthetics on opioid inhibition of calcium current in rat dorsal root ganglion neurons

Neuraxial analgesia is often provided using a mixture of local anesthetics and opioids. This combination of agents provides better pain relief and is generally associated with fewer side effects than when either drug is given alone. Local anesthetics have been shown to alter signaling of other G protein-coupled receptors, but little is known about their effect on opioid receptor signaling. Because opioids produce analgesia at least in part by inhibiting presynaptic Ca channels, we have evaluated the effects of tetracaine and bupivacaine on opioid-mediated inhibition of Ca channels in dorsal root ganglion neurons. The μ-opioid specific agonist DAMGO (1 μM) inhibited Ca channels in both the absence and presence of tetracaine (50 or 100 μM). However, the extent of DAMGO inhibition in the presence of both concentrations of tetracaine was less than that observed in the absence of tetracaine. DAMGO inhibition decreased from 39.2 ± 24.4% in control to 34.2 ± 24.4% with 50 μM tetracaine ( n = 16; p < 0.05), and from 40.5 ± 19.6% in control to 34.6 ± 20.5% with 100 μM tetracaine ( n = 10; p < 0.05). Similar results were seen with bupivacaine. Tetracaine also decreased the voltage-dependent facilitation of Ca channel currents when G proteins were activated by either DAMGO or the non-hydrolyzable GTP analogue (GTPγS), suggesting that tetracaine weakens the interaction between G protein βγ subunits and the Ca channel. Overall, these results suggest that local anesthetics decrease opioid inhibition of Ca channel activity by interfering with the GTP-mediated signal transduction between opioid receptors and Ca channels.

Calmodulin Kinase II Is Involved in Voltage-dependent Facilitation of the L-type Cav1.2 Calcium Channel: IDENTIFICATION OF THE PHOSPHORYLATION SITES

Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.

α&lt;sub&gt;1c&lt;/sub&gt;サブユニットとα&lt;sub&gt;1c&lt;/sub&gt;/βサブユニットからなるL型Caチャネルの定常状態不活性化と電位依存性電流増幅の区分

The L-type Ca2+ channel has a unique kinetic property known as voltage-dependent facilitation. Many researchers have repeatedly investigated the mechanism in response to the voltage-dependent facilitation since the first observation by Fenwick et al. in 1982. Electrophysiological evaluations of voltage-dependent facilitation, however, remain inconsistent, partially because of its unclear definition. Some scientists understand it as a current augmentation by a conditioning prepulse prior to the test pulse, and others understand it as a result of the U-shape steady-state inactivation curve. We therefore investigated to identify the distinction between the voltage-dependent facilitation and the steady-state inactivation, by use of Ba2+ as the charge in order to avoid the other inactivation mechanism or the Ca(2+)-dependent inactivation upon this analysis. Conventional whole-cell mode patch clamp technique was applied to chinese hamster fibroblast (CHW) cells that express the alpha1c subunit alone or the alpha1c subunit with the beta subunit (alpha1c/beta) derived from rabbit heart to investigate the voltage-dependent facilitation depending on the composition of the subunits. Coexpression of the beta subunit augmented alpha1 subunit channel current and shifted current-voltage relation towards hyperpolarized direction. In the experiment using conventional double pulse protocol to investigate steady-state inactivation, alpha1c subunit channel current and alpha1c/beta subunit channel current were not fully inactivated. Subtraction of the steady-state inactivation component from whole recovered current enabled us to identify the voltage-dependent facilitation component of the L-type Ca2+ channel. The voltage-dependent facilitation of the alpha1 subunit current and the alpha1c/beta subunit current were identical in kinetics, and could be generated at 0 mV or depolarized potentials partially overlapped with the potential range for the steady-state inactivation of the current. These results suggest that the voltage-dependent facilitation of the L-type Ca2+ channel could be formed by the alpha1c subunit without interaction with the beta subunit, and that the range for the voltage-dependent facilitation and the steady-state inactivation overlap each other at 0 mV or more depolarized potentials up to approximately + 100 mV.

Protein kinase A mediates voltage-dependent facilitation of Ca2+ current in presynaptic hair cells in Hermissenda crassicornis.

The simplest cellular model for classical conditioning in the nudibranch mollusk, Hermissenda crassicornis, involves the presynaptic hair cells and postsynaptic photoreceptors. Whereas the cellular mechanisms for postsynaptic photoreceptors have been studied extensively, the presynaptic mechanisms remain uncertain. Here, we determined the phenotype of the voltage-dependent Ca(2+) current in the presynaptic hair cells that may be directly involved in changes in synaptic efficacy during classical conditioning. The Ca(2+) current can be classified as a P-type current because its activation voltage under seawater recording conditions is approximately -30 mV, it showed slow inactivation, and it is reversibly blocked by omega-agatoxin-IVA. The steady-state activation and inactivation curves revealed a window current, and the single-channel conductance is approximately 20 pS. The P-type current was enhanced by cAMP analogs (approximately 1.3-fold), and by forskolin, an activator of adenylyl cyclase (approximately 1.25-fold). In addition, the P-type current showed voltage-dependent facilitation, which is mediated by protein kinase A (PKA). Specifically, the PKA inhibitor peptide [PKI(6-22)amide] blocked the enhancement of the Ca(2+) current produced by conditioning depolarization prepulses. Because neurotransmitter release is mediated by Ca(2+) influx via voltage-gated Ca(2+) channels, and because of the nonlinear relationship between the Ca(2+) influx and neurotransmitter release, we propose that voltage-dependent facilitation of the P-type current in hair cells would produce a robust change in synaptic efficacy.

Functional Interaction between Mouse Spermatogenic LVA and Thapsigargin-Modulated Calcium Channels

The acrosome reaction in mouse is triggered by a long-lasting calcium signaling produced by a chain of openings of several calcium channels, a low-voltage-activated (LVA) calcium channel, an inositol trisphosphate receptor (IP 3R), and the store-operated calcium channel TRP2. Since mature sperm cells are refractory to patch clamp experiments, we study the functional interactions among those sperm calcium channels in spermatogenic cells. We have studied the role of cytosolic calcium in voltage-dependent facilitation of low voltage-activated calcium channels. Calcium concentration was modified through the inclusion of the calcium buffers, EGTA and BAPTA, in the recording pipette solution, and by addition of calcium modulators like thapsigargin and the calcium ionophore A23187. We demonstrate that lowering calcium concentration below resting level allows to evidence a voltage-dependent facilitation. We also show that LVA calcium channels present strong voltage-dependent inhibition by thapsigargin. This effect is independent of cytosolic calcium elevation secondary to calcium store depletion and to the activation of TRP channels. Our data evidence an interesting functional relationship, in this cell type, between LVA channels and proteins whose activity is related to calcium filling state of the endoplasmic reticulum (presumably TRP channels and inositol triphosphate receptor). These relationships may contribute to the regulation of calcium signaling during acrosome reaction of mature sperm cell.

Voltage-independent Inhibition of P/Q-type Ca2+Channels in Adrenal Chromaffin Cells via a Neuronal Ca2+Sensor-1-dependent Pathway Involves Src Family Tyrosine Kinase

In common with many neurons, adrenal chromaffin cells possess distinct voltage-dependent and voltage-independent pathways for Ca(2+) channel regulation. In this study, the voltage-independent pathway was revealed by addition of naloxone and suramin to remove tonic blockade of Ca(2+) currents via opioid and purinergic receptors due to autocrine feedback inhibition. This pathway requires the Ca(2+)-binding protein neuronal calcium sensor-1 (NCS-1). The voltage-dependent pathway was pertussis toxin-sensitive, whereas the voltage-independent pathway was largely pertussis toxin-insensitive. Characterization of the voltage-independent inhibition of Ca(2+) currents revealed that it did not involve protein kinase C-dependent signaling pathways but did require the activity of a Src family tyrosine kinase. Two structurally distinct Src kinase inhibitors, 4-amino-5-(4-methylphenyl)7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP1) and a Src inhibitory peptide, increased the Ca(2+) currents, and no further increase in Ca(2+) currents was elicited by addition of naloxone and suramin. In addition, the Src-like kinase appeared to act in the same pathway as NCS-1. In contrast, addition of PP1 did not prevent a voltage-dependent facilitation elicited by a strong pre-pulse depolarization indicating that this pathway was independent of Src kinase activity. PPI no longer increased Ca(2+) currents after addition of the P/Q-type channel blocker omega-agatoxin TK. The alpha(1A) subunit of P/Q-type Ca(2+) channels was immunoprecipitated from chromaffin cell extracts and found to be phosphorylated in a PP1-sensitive manner by endogenous kinases in the immunoprecipitate. A high molecular mass (around 220 kDa) form of the alpha(1A) subunit was detected by anti-phosphotyrosine, suggesting a possible target for Src family kinase action. These data demonstrate a voltage-independent mechanism for autocrine inhibition of P/Q-type Ca(2+) channel currents in chromaffin cells that requires Src family kinase activity and suggests that this may be a widely distributed pathway for Ca(2+) channel regulation.

G-protein- and cAMP-dependent L-channel gating modulation: a manyfold system to control calcium entry in neurosecretory cells.

Voltage-gated Ca2+ channels are crucial to the control of Ca2+ entry in neurosecretory cells. In the chromaffin cells of adrenal medulla, paracrinally or autocrinally released neurotransmitters induce profound changes in Ca2+ channel gating and Ca2+-dependent events controlling catecholamine secretion and cell activity. The generally held view of these processes is that neurotransmitter-induced modulation of the most widely expressed Ca2+ channels in these cells (N-, P/Q- and L-type) follows two distinct pathways: a direct membrane-delimited Gi/o-protein-induced inhibition of N- and P/Q-type and a remote cAMP-mediated facilitation of L-channels. Both actions depend on voltage, although with remarkably different molecular and kinetic aspects. Recent findings, however, challenge this simple scheme and suggest that L-channels do not require strong pre-pulses to be recruited or facilitated. They are available during normal depolarizations and may be tonically inhibited by Gi/o proteins activated by the released neurotransmitters. Like the N- and P/Q-channels, this autocrine modulation is localized to membrane microareas. Unlike N- and P/Q-channels, however, the inhibition of L-channels is largely independent of voltage and develops in parallel with cAMP-mediated potentiation of channel gating. As L-channels play a crucial role in the control of catecholamine release in chromaffin cells, the two opposite modulations mediated by Gi/o proteins and cAMP may represent an effective way to broaden the dynamic range of Ca2+ signals controlling exocytosis. Here, we review the basic features of this novel L-type channel inhibition comparing it to the well-established forms of L-channel potentiation and voltage-dependent facilitation.

Multiple structural elements contribute to voltage-dependent facilitation of neuronal α 1C (Ca V1.2) L-type calcium channels

Voltage- and frequency-dependent facilitation of calcium channel activity has been implicated in a number of key physiological processes. Various mechanisms have been proposed to mediate these regulations, including a switch between channel gating modes, voltage-dependent phosphorylation, and a voltage-dependent deinhibition of G-protein block. Studying such modulation on recombinant Ca channels expressed in oocytes, we previously reported that α 1C L-type calcium channel contrast with non-L type Ca channels by its ability to exhibit facilitation by pre-depolarization (Voltage-dependent facilitation of a neuronal α IC L-type calcium channel, E. Bourinet et al., EMBO Journal, 1994; 13, 5032–5039). To further analyze this effect, we have investigated the molecular determinants which mediate the differences in voltage-dependent facilitation between «facilitable» α 1C and «non facilitable» α 1E calcium channels. We used a series of chimeras which combine the four transmembrane domains of the two channels. Results show that the four domains of α 1C contribute to facilitation, with domain I being most critical. This domain is required but not sufficient alone to generate facilitation. The minimal requirement to observe the effect is the presence of domain I plus one of the three others. We conclude that similarly to activation gating, voltage-dependent facilitation of α 1C is a complex process which involves multiple structural elements were domains I and III play the major role.

Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Ca(v)beta subunits.

N-type and P/Q-type Ca(2+) channels are inhibited by neurotransmitters acting through G protein-coupled receptors in a membrane-delimited pathway involving Gbetagamma subunits. Inhibition is caused by a shift from an easily activated "willing" (W) state to a more-difficult-to-activate "reluctant" (R) state. This inhibition can be reversed by strong depolarization, resulting in prepulse facilitation, or by protein kinase C (PKC) phosphorylation. Comparison of regulation of N-type Ca(2+) channels containing Cav2.2a alpha(1) subunits and P/Q-type Ca(2+) channels containing Ca(v)2.1 alpha(1) subunits revealed substantial differences. In the absence of G protein modulation, Ca(v)2.1 channels containing Ca(v)beta subunits were tonically in the W state, whereas Ca(v)2.1 channels without beta subunits and Ca(v)2.2a channels with beta subunits were tonically in the R state. Both Ca(v)2.1 and Ca(v)2.2a channels could be shifted back toward the W state by strong depolarization or PKC phosphorylation. Our results show that the R state and its modulation by prepulse facilitation, PKC phosphorylation, and Ca(v)beta subunits are intrinsic properties of the Ca(2+) channel itself in the absence of G protein modulation. A common allosteric model of G protein modulation of Ca(2+)-channel activity incorporating an intrinsic equilibrium between the W and R states of the alpha(1) subunits and modulation of that equilibrium by G proteins, Ca(v)beta subunits, membrane depolarization, and phosphorylation by PKC accommodates our findings. Such regulation will modulate transmission at synapses that use N-type and P/Q-type Ca(2+) channels to initiate neurotransmitter release.

Roles of alpha(1) and alpha(1)/beta subunits derived from cardiac L-type Ca(2+) channels on voltage-dependent facilitation mechanisms.

Strong depolarization pulses facilitate L-type Ca(2+) channels in various cell types including cardiac myocytes. The mechanisms underlying prepulse facilitation are controversial with respect to the requirements for channel subunits, cAMP-dependent protein kinase, and additional anchor proteins. The properties of voltage-dependent facilitation of the L-type Ca(2+) channel was studied in recombinant cardiac alpha(1) subunits with or without cardiac beta subunit, expressed in Chinese hamster fibroblast cells. The magnitude of voltage-dependent I(Ba) facilitation in the alpha(1) subunit channel is dependent on the duration of the prepulse as well as on the interval duration between prepulse and test pulse. The characteristics of this facilitation were not affected by coexpression of the beta subunit. These results indicate that cardiac alpha(1) subunits exhibit voltage-dependent facilitation because of their own intrinsic structure, independent of any other accessory subunit or additional regulatory proteins, and that cardiac beta subunits have no essential regulatory role at the onset or continuance of the voltage-dependent facilitation.