Molecular and pharmacological bases for the gating regulation of L-type voltage-dependent Ca2+ channels

The voltage-dependent L-type Ca(2+) channel plays a key role in the spacial and temporal regulation of Ca(2+). In cardiac excitation-contraction coupling, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), triggered by Ca(2+) entry through the nearby L-type Ca(2+) channel, induces the Ca(2+)-dependent inactivation (CDI) of the Ca(2+) channel. We demonstrated that the CICR-dependent CDI of L-type Ca(2+) channels, under control of the privileged cross-signaling between L-type Ca(2+) channels and RyRs, plays important roles for monitoring and tuning the SR Ca(2+) content via changes of AP waveform and the amount of Ca(2+)-influx during AP in ventricular myocytes. L-type Ca(2+) channels are modulated by the binding of Ca(2+) channel antagonists and agonists to the pore-forming alpha(1C) subunit. We identified Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region of the alpha(1C) subunit as critical determinants of the binding of dihydropyridines (DHP). Interestingly, double mutant Ca(2+) channel (F1112A/S1115A) failed to discriminate between a DHP Ca(2+) channel agonist and antagonist stereoisomers. We proposed that Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region is required for the stabilization of the Ca(2+) channel in the open state by Ca(2+) channel agonists and further proposed a novel model for the DHP-binding pocket of the alpha(1C) subunit. These integrative studies on the gating regulation of cardiac L-type Ca(2+) channels will provide the molecular basis for the pharmacology of Ca(2+) channel modulators.     

Possible involvement of both N- and L-type voltage-dependent Ca channels in adrenergic neurotransmission of canine saphenous veins in low Ca2+ plus tetraethylammonium medium

Summary The involvement of N- and L-type voltage-dependent Ca channels (VDCCs) in adrenergic neurotransmission under the superfusion with 0.25 mM Ca²⁺ + 20 mM tetraethylammonium (low Ca²⁺ + TEA) medium has been studied by examining the effects of -conotoxin GVIA (-CTX) and dihydropyridine antagonists and agonist on transmural nerve stimulation (TNS)-evoked ³H overflow from canine saphenous veins preloaded with [³H]-noradrenaline. Nisoldipine (10 and 30 M) and nifedipine (30 M) reduced significantly the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium, while the two dihydropyridine antagonists failed to suppress it in normal Krebs medium. Bay K 8644 (30 and 100 nM) produced a significant and concentration-dependent enhancement of the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium. The enhancing effects of Bay K 8644 were antagonized by both 3 M nisoldipine and 10 tM nifedipine. -CTX inhibited markedly the TNS-evoked ³H overflow in both normal Krebs and low Ca²⁺ + TEA media, the inhibition by -CTX being ten times more potent in low Ca²⁺ + TEA medium. Nisoldipine (30 M), when combined with 1 nM -CTX, produced a further significant inhibition of the TNS-evoked ³H overflow in low Ca²⁺ + TEA medium. However, no additional inhibition by 30 M nisoldipine was observed when -CTX concentration was raised to 2 nM. In the veins superfused with normal Krebs medium, nisoldipine (30 M) did not affect the inhibitory effect of 10 nM -CTX on the evoked ³H overflow. The low Ca²⁺ + TEA medium increased the spontaneous ³H overflow, which was not influenced by -CTX and dihydropyridines. These results suggest that in low Ca²⁺ + TEA medium but not normal Krebs one, Ca²⁺ entry via both N- and L-type VDCCs may be involved in adrenergic neurotransmission in the canine saphenous veins. Correspondence to Y. Takata at the above address     

Use of transgenic mice to study voltage-dependent Ca2+ channels

During the past decade a great number of genes encoding high- and low-voltage-dependent Ca(2+) channels and their accessory subunits have been cloned. Studies of Ca(2+) channel structure-function relationships and channel regulation using cDNA expression in heterologous expression systems have revealed intricate details of subunit interaction, regulation of channels by protein kinase A (PKA) and protein kinase C (PKC), drug binding sites, mechanisms of drug action, the ion conduction pathway and other aspects of channel function. In recent years, however, we have arrived at the brink of an entirely new strategy to study Ca(2+) channels by overexpressing or knocking out genes encoding these channels in transgenic mice. In this article, various models of gene knockout or gene overexpression will be discussed. This new approach will reveal many secrets regarding Ca(2+) channel regulation and the control of Ca(2+)-dependent cellular processes.     

Ginsenoside Rg1 protects neurons from hypoxic-ischemic injury possibly by inhibiting Ca2+ influx through NMDA receptors and L-type voltage-dependent Ca2+ channels

The purpose of this study is to assess the neuroprotective effect of Rg1, a ginsenoside. We measured cell viability and lactate dehydrogenase (LDH) release from primary culture of rat hippocampal neurons and electrical activities in hippocampal slices of rats, before and after the neurons were deprived of oxygen and glucose. In addition, cerebral damage was evaluated with magnetic resonance imaging after middle cerebral artery was occluded transiently. Nissl staining was used for histological observation and immunohistochemistry analysis for activated caspase-3 expression of the brain. Furthermore, calcium influx was measured with laser confocal microscopy in neurons perfused with KCl (50 mM) or N-methyl-d-aspartate (NMDA, 1 mM), or deprived of oxygen and glucose. The influences of ginsenoside Rg1 on these parameters were determined simultaneously. We found that treatment of Rg1: 1) increased the neuronal viability; 2) promoted the recovery of electrical activity in hippocampal slices; 3) reduced the release of LDH, cerebral damage area, neuronal loss and expression of caspase-3; and 4) inhibited calcium influx induced by NMDA, KCl or oxygen/glucose deprivation. However, the protective effect of Rg1 was blocked by mifepristone, an antagonist of glucocorticoid receptors. Taken together, these results suggest that ginsenoside Rg1 can reduce neuronal death, including apoptotic cell death, induced by hypoxic-ischemic insults. This neuroprotective effect is probably mediated by the activation of glucocorticoid receptors, and by the inhibition of calcium influx through NMDA receptors and L-type voltage-dependent Ca2+ channels and the resultant reduction of intracellular free Ca2+.     

Tributyltin-induced Ca(2+) mobilization via L-type voltage-dependent Ca(2+) channels in PC12 cells

The effects of tributyltin (TBT) on cytosolic Ca(2+) concentration ([Ca(2+)](c)) and cell viability were investigated in nerve growth factor-differentiated PC12 cells. TBT concentration dependently increased [Ca(2+)](c) with an EC(50) value of 0.07μM. This effect was markedly reduced by removal of the extracellular Ca(2+) or membrane depolarization with a high K(+) medium, but unaffected by thapsigargin causing depletion of intracellular Ca(2+) stores. The L-type voltage-dependent Ca(2+) channel (VDCC) blocker nicardipine blocked the effect of TBT, but the N-type VDCC blocker ω-conotoxin did not. TBT decreased the number of viable cells with an EC(50) value of 0.09μM. The TBT-induced cell death was prevented by nicardipine or by chelating the cytosolic Ca(2+) with BAPTA-AM, but not by ω-conotoxin. The results show that TBT causes an increase in [Ca(2+)](c) via activating L-type VDCCs, and support the idea that the organotin-induced cell death arises through Ca(2+) mobilization via L-type VDCCs.     

The beta 1 subunit of L-type voltage-gated Ca2+ channels independently binds to and inhibits the gating of large-conductance Ca2+-activated K+ channels

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels encoded by the Slo1 gene are ubiquitously expressed, and they play a role in regulation of many cell types. In excitable cells, BK(Ca) channels and voltage-activated Ca(2+) channels often form functional complexes that allow the cytoplasmic domains of BK(Ca) channels to lie within spatially discrete calcium microdomains. Here, we report a novel protein interaction between the beta1-subunit of L-type voltage-activated calcium channels (Ca(v)beta1) and critical regulatory domains of Slo1 that can occur in the absence of other proteins. This interaction was identified by a yeast two-hybrid screen, and it was confirmed by confocal microscopy in native neurons, by coimmunoprecipitation, and by direct binding assays. The Ca(v)beta1 subunit binds within the calcium bowl domain of Slo1 that mediates a portion of high-affinity Ca(2+) binding to BK(Ca) channels and also to a noncanonical Src homology 3 (SH3) domain-binding motif within Slo1. Binding of Ca(v)beta1 markedly slows Slo1 activation kinetics, and it causes a significant decrease in Ca(2+) sensitivity in inside-out and in dialyzed cells, even in the absence of pore-forming subunits of voltage-gated Ca(2+) channels. The guanylate kinase domain of Ca(v)beta1 mediates Slo1 regulation through its binding to calcium bowl domains, and this domain of Ca(v)beta1 is necessary and sufficient for the observed effects on BK(Ca) activation. Binding of Ca(v)beta1 to SH3-binding motifs may stabilize the interaction with Slo1, or it may contribute to formation of other complexes, but it does not seem to affect Ca(2+)-dependent gating of Slo1. Binding of Ca(v)beta1 does not affect cell surface expression of Slo1 in human embryonic kidney 293T cells.     

Negative modulation of L-type Ca2+ channels via beta-adrenoceptor stimulation in guinea-pig detrusor smooth muscle cells

beta-Adrenergic stimulation enhances the activity of L-type Ca(2+) channels through mechanisms mediated by adenosine 3'5'-cyclic monophosphate (cAMP) and protein kinase A in cardiac myocytes. However, in smooth muscle cells, the effect of beta-adrenoceptor stimulation on the L-type Ca(2+) channel activity has been controversial, and the exact mechanism is still unclear. The present study was aimed at elucidating the effect of beta-adrenergic stimulation upon the activity of L-type Ca(2+) channels in guinea-pig detrusor smooth muscle cells. Isoproterenol (0.1-1 microM) inhibited Ba(2+) currents through L-type Ca(2+) channels (I(Ba)). Isoproterenol (0.1 microM) shifted the steady-state inactivation curve to negative voltages by 11 mV without affecting activation curves. The stimulation of cAMP-mediated signal transduction pathway by forskolin, 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP), or the intracellular application of cAMP also mimicked the effects of isoproterenol on I(Ba), which was blocked by the inhibition of protein kinase A. These results indicate that, in detrusor smooth muscles, the stimulation of beta-adrenoceptors exerts negative modulation of L-type Ca(2+) channels via cAMP/protein kinase A-dependent mechanism.     

Interaction of three structurally distinct Ca2+ channel activators with single L-type Ca2+ channels

The dihydropyridine S(-)-Bay K 8644 (Bay K), the benzoylpyrrole FPL 64176 (FPL) and the benzodiazocine CGP 48506 (CGP) are structurally unrelated L-type Ca2+ channels agonists. The aim of our study was to investigate whether these three drugs interact with different binding sites and thereby modulate the behaviour of L-type Ca2+ channels in a qualitatively different manner. Single-channel recordings were performed on CHO cells stably expressing the alpha1C-b subunit of the L-type Ca2+ channel. Mean open time and open probability were determined sweep by sweep and the effects of CGP (10(-4) M), Bay K (10(-6) M) and FPL (10(-6) M) were compared. All three compounds increased mean open time and open probability when applied alone. However, the gating pattern changes induced by each drug were qualitatively and quantitatively different. We also applied binary mixtures and analysed the resulting sweeps with respect to their gating pattern. The application of mixtures did result in a gating pattern not seen with any of the single drugs. The mixture of CGP and FPL led to a prolonged mean open time compared with each single drug. The mixture of Bay K and FPL exhibited an open probability lower than with each single drug. The mixture of CGP and Bay K increased the mean open time per sweep like Bay K, but the number of openings was similar to the level seen with CGP alone. These results cannot be explained by assuming alternative binding of the drugs to a single binding site. We therefore conclude that Bay K, CGP and FPL bind to different but interacting sites on the L-type Ca2+ channel.     

Mefenamic acid as a novel activator of L-type voltage-dependent Ca2+ channels in smooth muscle cells from pig proximal urethra

The effects of mefenamic acid and Bay K 8644 on voltage-dependent nifedipine-sensitive inward Ba2+ currents in pig urethral myocytes were investigated by use of conventional whole-cell configuration patch clamp. Mefenamic acid increased the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba2+ current without shifting the position of the current-voltage relationship. Mefenamic acid (300 microM) caused little shift in the activation curve although the voltage dependence of the steady-state inactivation was shifted to more positive potentials by 11 mV in the presence of mefenamic acid. Bay K 8644 (> or = 100 nM) enhanced voltage-dependent nifedipine-sensitive inward Ba2+ currents in a concentration- and voltage-dependent manner, shifting the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction. Bay K 8644 (1 microM) significantly shifted the voltage dependence of the activation curve to more negative potentials by approximately 9 mV although Bay K 8644 caused little shift in the steady-state inactivation curve. These results indicate that mefenamic acid increased voltage-dependent nifedipine-sensitive inward Ba2+ currents through the activation of L-type Ca2+ channels with different kinetics from those of Bay K 8644 in pig urethral myocytes.     

Voltage-dependence of Ca2+ agonist effect of YC-170 on cardiac L-type Ca2+ channels.

1. We investigated the voltage-dependence of the agonist actions of YC-170, a dihydropyridine (DHP) derivative, on cardiac L-type Ca2+ channels in rabbit ventricular cells, using the patch clamp technique. The characteristics of YC-170 were compared with those of other DHP Ca2+ agonists (Bay K 8644, CGP 28392). Ca2+ channel activities were elicited by depolarizing pulses to 0 mV from a holding potential (HP) of either -80 mV or -40 mV. 2. YC-170 (10 microM) increased Ca2+ channel activities when HP was set at -80 mV. However, decreasing HP to -40 mV abolished the agonist action. The agonist effect of Bay K 8644 (1 microM) on Ca2+ channels was elicited to the same extent at either HP. CGP 28392 (10 microM) also increased Ca2+ channel activities at both HPs, but its agonist effect was significantly larger at an HP of -80 mV than at -40 mV. 3. All of the three DHP Ca2+agonists prolonged open times of Ca2+ channels, but the prolongation did not correspond to the voltage-dependence of Ca2+ agonist effects of the three DHPs. 4. YC-170 markedly reduced the closed time of the Ca2+ channel when the HP was at -80 mV, but prolonged it at HP of -40 mV. Bay K 8644 reduced closed times at an HP of -80 mV. At an HP of -40 mV, Bay K 8644 slightly reduced closed times. CGP 28392 reduced closed times at an HP of -80 mV and prolonged those at an HP of -40 mV. Thus the voltage-dependence of the agonist effects of these agents was in good agreement with the voltage-dependence of changes in closed times of Ca2+ channel. 5. Two mechanisms may be involved in the agonist action of YC-170; a prolongation of open times, and a reduction of closed times of Ca2+ channels, i.e. an increase in reopening. The former mechanism is not dependent on Hp and the latter mechanism is highly dependent on HP. Thus, the voltage-dependence of the agonist action may be attributed to the voltage-dependence of their enhancing effect on reopening of Ca2+ channels.     

Receptor-mediated modulation of voltage-dependent Ca2+ channels via heterotrimeric G-proteins in neurons

The activity of voltage-dependent Ca2+ channels is highly regulated by neurotransmitter receptors coupled to heterotrimeric G-proteins. In the expression studies using cloned Ca2+ channel subunits, it has been clarified that the main mechanism of the inhibition of N-type channel current is mediated directly by G-protein betagamma subunits in a membrane-delimited and voltage-sensitive manner. In addition, recent studies have also clarified that N-type channels are modulated by several G-protein alpha subunits in different ways. Among them, G(alpha o) mediates a voltage-resistant inhibition of N-type current by neurotransmitters. This type of inhibition is more apparent in the case of P/Q-type channels in both native cells and expression systems. Moreover, other G-protein subunits, such as G(alpha q) and G(alpha s), also seem to regulate N-type channels in a membrane-delimited manner. The fine tunings of Ca2+ channel activity by intracellular proteins have physiological and pathological meanings in the regulation of Ca2+ influx into excitable cells by neurotransmitters and pharmacological implications as novel drug targets for controlling Ca2+ influx.     

Acidosis-induced protein tyrosine phosphorylation depends on Ca2+ influx via voltage-dependent Ca2+ channels in SHR aorta

The contractile response to acidosis in isolated aorta from spontaneously hypertensive rat (SHR) depends upon tyrosine phosphorylation of phosphatidylinositol 3 kinase (PI3-kinase) and Ca2+ influx via voltage-dependent Ca2+ channels (VDCC). In this study, verapamil, a VDCC inhibitor, was shown to markedly inhibit acidic pH-induced contraction, whereas the residual contraction in the presence of verapamil was unaffected by the PI3-kinase inhibitor, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY-294002). Interestingly, the LY-294002-insensitive component of contraction was further inhibited by verapamil in the presence of LY-294002. Western blotting revealed that acidosis stimulated tyrosine phosphorylation of p85, which was abolished when tissues were pretreated with tyrphostin 23, a tyrosine kinase inhibitor, verapamil or EGTA. In fura-2-loaded aortic strips, acidosis induced a rise in intracellular Ca2+ ([Ca2+]i) that was partially inhibited by LY-294002. The residual increase in [Ca2+]i caused by acidosis in the presence of LY-294002 was abolished by verapamil. These findings suggest that acidosis-induced Ca2+ influx through VDCC is the upstream event leading to the tyrosine phosphorylation of PI3-kinase, which in turn contributes to the enhancement of Ca2+ entry to some extent in SHR aorta.     

The effects of flavoxate hydrochloride on voltage-dependent L-type Ca2+ currents in human urinary bladder

The effects of flavoxate hydrochloride (Bladderon, piperidinoethyl-3-methylflavone-8-carboxylate; hereafter referred as flavoxate) on voltage-dependent nifedipine-sensitive inward Ba(2+) currents in human detrusor myocytes were investigated using a conventional whole-cell patch-clamp. Tension measurement was also performed to study the effects of flavoxate on K(+)-induced contraction in human urinary bladder. Flavoxate caused a concentration-dependent reduction of the K(+)-induced contraction of human urinary bladder. In human detrusor myocytes, flavoxate inhibited the peak amplitude of voltage-dependent nifedipine-sensitive inward Ba(2+) currents in a voltage- and concentration-dependent manner (K(i) = 10 microM), and shifted the steady-state inactivation curve of Ba(2+) currents to the left at a holding potential of -90 mV. Immunohistochemical studies indicated the presence of the alpha(1C) subunit protein, which is a constituent of human L-type Ca(2+) channels (Ca(V)1.2), in the bundles of human detrusor smooth muscle. These results suggest that flavoxate caused muscle relaxation through the inhibition of L-type Ca(2+) channels in human detrusor.     

Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels

The effect of the Na+/Ca(2+)-exchange inhibitor KB-R7943 was investigated in spinal cord dorsal column ischemia in vitro. Oxygen/glucose deprivation at 37 degrees C for 1 h causes severe injury even in the absence of external Ca2+. KB-R7943 was very protective in the presence and absence of external Ca2+ implicating mechanisms in addition to extracellular Ca2+ influx through Na+/Ca(2+)-exchange, such as activation of ryanodine receptors by L-type Ca2+ channels. Indeed, blockade of L-type Ca2+ by nimodipine confers a certain degree of protection of dorsal column against ischemia; combined application of nimodipine and KB-R7943 was not additive suggesting that KB-R7943 may also act on Ca2+ channels. KB-R7943 reduced inward Ba2+ current with IC50 = 7 microM in tsA-201 cells expressing Ca(v)1.2. Moreover, nifedipine and KB-R7943 both reduced depolarization-induced [Ca2+]i increases in forebrain neurons and effects were not additive. Nimodipine or KB-R7943 also reduced ischemic axoplasmic Ca2+ increase, which persisted in 0Ca2+/EGTA perfusate in dorsal column during ischemia. While KB-R7943 cannot be considered to be a specific Na+/Ca2+ exchange inhibitor, its profile makes it a very useful neuroprotectant in dorsal columns by: reducing Ca2+ import through reverse Na+/Ca2+ exchange; reducing influx through L-type Ca2+ channels, and indirectly inhibiting Ca2+ release from the ER through activation of ryanodine receptors.     

Effects of raloxifene on voltage-dependent T-type Ca2+ channels in mouse spermatogenic cells

Voltage-dependent T-type Ca(2+) channels have eminent roles in sperm function. In the present study, we investigated the effects of raloxifene, a selective estrogen receptor modulator, on T-type Ca(2+) channels in mouse spermatogenic cells by using both electrophysiological and molecular techniques. We found that T-type calcium currents (I(T-Ca)) were inhibited by raloxifene in a concentration-dependent manner with an IC(50) of 2.97 μM, as assessed with the patch clamp technique. Application of raloxifene at 2 μM inhibited I(T-Ca) by 54.9 ± 2.1% at -20 mV (n = 10, p < 0.05). Furthermore, raloxifene-induced inhibition of I(T-Ca) was associated with a negative shift of both the activation and the steady-state inactivation properties. The time constants of activation and inactivation were decreased, the time constant of deactivation was increased, but the time constant of recovery was not affected. In addition, the inhibitory effects of raloxifene and 17β-estradiol on I(T-Ca) were unaffected by the estrogen receptor antagonist ICI 182,780. We also found that raloxifene treatment decreased the mRNA expression of Ca(V)3.2 and Ca(V)3.3, but not Ca(V)3.1 in GC-2spd (ts) cells (mouse spermatocyte cell line), as assessed by real-time RT-PCR. Taken together, these data indicate that in mouse spermatogenic cells, raloxifene decreases I(T-Ca) independent of classical estrogen, and the mRNA expression of T-type calcium channels and therefore may affect male reproductive function.     

Inhibition of voltage-dependent Ca2+ channels of porcine tracheal smooth muscle by the novel Ca2+ channel antagonist RWJ-22108

• 1.1. We compared electrophysiological effects of the bronchoselective Ca²⁺ channel antagonist RWJ-22108 on voltage-dependent Ca²⁺ channels (VDCs) of porcine tracheal smooth muscle cells to the effects of nicardipine and verapamil.
• 2.2. Each of the three Ca²⁺ channel antagonists tested inhibited inward Ca²⁺ currents (I_Ca) measured by whole-cell patch clamp techniques. Inhibition was dose-dependent with ∼50% inhibition of peak I_Ca at +20 mV obtained with 3 × 10⁻⁶M RWJ-22108, 3 × 10⁻⁷M nicardipine, or 10⁻⁵M verapamil.
• 3.3. Both RWJ-22108 (3 × 10⁻⁶M) and nicardipine (3 × 10⁻⁷M) shifted the voltage dependence of steady-state inactivation to more negative potentials; however, the change in the potential of half-maximal inactivation induced by RWJ-22108 (−18 mV) was significantly greater than that induced by nicardipine (−12 mV). Verapamil did not alter the voltage dependence of inactivation.
• 4.4. We conclude that inhibition of VDCs by RWJ-22108 is qualitatively similar to that by nicardipine but with a greater stabilizing effect on the inactivated channel state.

     

Triphenyltin-induced increase in the intracellular Ca2+ of dissociated mammalian CNS neuron: its independence from voltage-dependent Ca2+ channels.

To test the possibility that triphenyltin (TPT) increases the intracellular Ca2+ ([Ca2+]i) in neurons as found previously in thymocytes, the effect of TPT on [Ca2+]i was examined in rat cerebellar neurons by a flow-cytometer with fluorescent dyes. TPT at concentrations ranging from 3 x 10(-7) M to 1 x 10(-5) M dose-dependently increased the [Ca2+]i. The TPT-induced increase in [Ca2+]i was not attenuated by a Ca2+ channel blocker, suggesting that it was not dependent on voltage-dependent Ca2+ channels. As the concentration of external Ca2+ ([Ca2+]e) increased, TPT produced a more profound increase in the [Ca2+]i. However, the increase in the [Ca2+]i by TPT was observed even in nominally [Ca2+]e-free solution. These results suggest two possibilities. First, TPT may promote Ca(2+)-influx to the neuron. Secondly, TPT may affect the intracellular Ca-store sites. This study is relevant to the neurotoxicity of organotins because it has become progressively clear that sustained increases in the [Ca2+]i can activate various Ca(2+)-dependent degradative processes.     

Kinetic effects of FPL 64176 on L-type Ca2+ channels in cardiac myocytes

To characterize the effects of the Ca2+ channel agonist FPL 64176 on L-type Ca2+ current in isolated rat ventricular myocytes, certain of its effects were compared with those of a better known agonist, S (-) Bay K 8644. Both drugs enhance currents elicited by depolarizing pulses and enhance and slow the decay of tail currents elicited by subsequent repolarization. Both drugs shift the voltage dependence of activation and of inactivation approximately 10 mV in the negative direction, but FPL 64176 slows the rate of both activation and the decline of Ca2+ current during a depolarization, whereas Bay K 8644 accelerates the rate of current decay under the same conditions. In single channel studies in on-cell recording mode, FPL 64176 produced a great lengthening of the channel open time, produced very long openings when the channels were repolarized after a depolarizing stimulus, and had only modest effects on mean closed times and on first latency distributions. FPL 64176 and Bay K 8644 also had minimal effects on L-type channel "on" gating currents, while the "off" gating currents were slowed, particularly at positive potentials. However, the effects on gating currents were too small to account for the prolonged tails observed in FPL 64176. Once the channel is open, FPL 64176 slows transitions to closed or inactivated channel states.     

Multiple structural elements contribute to voltage-dependent facilitation of neuronal alpha 1C (CaV1.2) 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 alpha(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 alpha(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" alpha(1C) and "non facilitable" alpha(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 alpha(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 alpha(1C) is a complex process which involves multiple structural elements were domains I and III play the major role.