Altered cocaine effects in mice lacking Ca(v)2.3 (alpha(1E)) calcium channel

Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Ca(v)2.3 (alpha(1E)) calcium channel to cocaine effects using Ca(v)2.3 knockout mice (Ca(v)2.3-/-). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Ca(v)2.3+/+), but failed to produce any response in Ca(v)2.3-/- mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Ca(v)2.3+/+ mice; however, it had no significant effect in Ca(v)2.3-/- mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Ca(v)2.3-/- mice. These data indicate that Ca(v)2.3 channel contributes to the locomotor-stimulating effect of cocaine, and the deletion of Ca(v)2.3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist.     

Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.     

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.     

T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.     

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.     

Differential expression of voltage-gated Ca(2+)-currents in cultivated aortic myocytes.

The expression of different types of Ca(2+)-channels was studied using the whole-cell patch-clamp technique in cultured rat aortic smooth-muscle myocytes. Ca(2+)-currents were identified as either low- or high voltage-activated (ICa,LVA or ICa,HVA, respectively) based on their distinct voltage-dependences of activation and inactivation, decay kinetics using Ba2+ as the charge carrier and sensitivity to dihydropyridines. The heterogeneity in the functional expression of the two types of Ca(2+)-channels in the cultured myocytes delineated four distinct phenotypes; (i), cells exhibiting only LVA currents; (ii), cells exhibiting only HVA currents; (iii), cells exhibiting both LVA and HVA currents and (iv), cells exhibiting no current. The myocytes exclusively expressed HVA currents both during the first five days in primary culture and after the cells had reached confluence (> 15 days). In contrast, LVA currents were expressed transiently between 5 and 15 days, during which time the cells were proliferating and had transient loss of contractility. Thus, both LVA and HVA Ca(2+)-current types contribute to Ca(2+)-signalling in cultured rat aortic myocytes. However, the differential expression of the two Ca2+ current types associated with differences in contractile and proliferative phenotypes suggest that they serve distinct cellular functions. Our results are consistent with the idea that LVA current expression is important for cell proliferation.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

Ca(2+) sensors of L-type Ca(2+) channel

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.     

Properties of voltage-gated Ca(2+) channels in rabbit ventricular myocytes expressing Ca(2+) channel alpha(1E) cDNA

Using the whole-cell patch-clamp technique, we have studied the properties of alpha(1E) Ca(2+) channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current (I(Ca,L)). Expression of green fluorescent protein significantly decreased the I(Ca,L). By contrast, expression of alpha(1E) with beta(2b) and alpha(2)/delta significantly increased the total Ca(2+) current, and in these cells a Ca(2+) antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni(2+) and was little affected by changing the charge carrier from Ca(2+) to Ba(2+) or by beta-adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of alpha(1E) did not generate T-like current in cardiac myocytes. On the other hand, expression of alpha(1E) decreased I(Ca,L) and slowed the I(Ca,L) inactivation. This inactivation slowing was attenuated by the beta(2b) coexpression, suggesting that the alpha(1E) may slow the inactivation of I(Ca,L) by scrambling with alpha(1C) for intrinsic auxiliary beta.     

Intracellular Ca(2+) channel immunoreactivity in neuroendocrine axon terminals

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca(2+) channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the alpha(1)1.3, alpha(1)2.1, alpha(1)2.2, and alpha(1)2.3 Ca(2+) channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca(2+) channels are dispersed. This contrasts with the highly punctate alpha(1)2.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca(2+) channel types. Ca(2+) channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.     

Classification of voltage-dependent Ca2+ channels in trigeminal ganglion neurons from neonatal rats

We examined the subtypes and characteristics of the Ca(2+) channel in small (diameter < 30 microm) trigeminal ganglion (TG) neurons from neonatal rats by means of whole cell patch clamp techniques. There were two current components, low-voltage activated (LVA) and high-voltage activated (HVA) I(Ba), with different activation ranges and waveforms. LVA I(Ba) elicited from a depolarizing step pulse at a holding potential (HP) of -80 mV was inhibited by 0.25 mM amiloride (62%), which did not produce any significant inhibition of the peak amplitude of HVA I(Ba). The application of 0.5 mM amiloride inhibited 10% of the HVA I(Ba). The LVA I(Ba) was also reduced by changing the HP from -80 to -60 mV (61%), and under these conditions the peak amplitude of HVA I(Ba) did not change significantly. In addition, HVA I(Ba) and LVA I(Ba) showed marked differences in their inactivation properties. Experiments with several Ca(2+) channel blockers revealed that on average, 26% of the HVA I(Ba) was nifedipine (10 microM) sensitive, 55% was sensitive to omega-conotoxinGVIA (1 microM), 4% was blocked by omega-agatoxinIVA (1 microM), and the remainder of the current that was resistant to the co-application of all three Ca(2+) channel blockers was 15% of the total current. These results suggest that the application of amiloride and the alteration of the holding potential level can discriminate between HVA and LVA Ba(2+) currents in TG neurons, and that TG neurons expressed T-, L-, N-, P-/Q- and R-type Ca(2+) channels.     

Effects of ginsenosides and their metabolites on voltage-dependent Ca(2+) channel subtypes

In previous reports we demonstrated that ginsenosides, active ingredients of Panax ginseng, affect some subsets of voltage-dependent Ca(2+) channels in neuronal cells expressed in Xenopus laevis oocytes. However, the major component(s) of ginseng that affect cloned Ca(2+) channel subtypes such as alpha(1C) (L)-, alpha(1B) (N)-, alpha(1A) (P/Q)-, a1E (R)- and a1G (T) have not been identified. Here, we used the two-microelectrode volt-age clamp technique to characterize the effects of ginsenosides and ginsenoside metabolites on Ba(2+) currents (IBa) in Xenopus oocytes expressing five different Ca(2+) channel subtypes. Exposure to ginseng total saponins (GTS) induced voltage-dependent, dose-dependent and reversible inhibition of the five channel subtypes, with particularly strong inhibition of the a1G-type. Of the various ginsenosides, Rb(1), Rc, Re, Rf, Rg(1), Rg(3), and Rh(2), ginsenoside Rg(3) also inhibited all five channel subtypes and ginsenoside Rh(2) had most effect on the a1C- and a1E-type Ca(2+) channels. Compound K (CK), a protopanaxadiol ginsenoside metabolite, strongly inhibited only the a(1G)-type of Ca(2+) channel, whereas M4, a protopanaxatriol ginsenoside metabolite, had almost no effect on any of the channels. Rg(3), Rh(2), and CK shifted the steady-state activation curves but not the inactivation curves in the depolarizing direction in the alpha(1B)- and alpha(1A)-types. These results reveal that Rg(3), Rh(2) and CK are the major inhibitors of Ca(2+) channels in Panax ginseng, and that they show some Ca(2+) channel selectivity.     

Retinoic acid reduces the cytotoxicity of cyclopentenyl cytosine in neuroblastoma cells

To know the function of the Ca2+ channel containing alpha(1)2.3 (alpha1E) subunit (Ca(v)2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Ca(v)2.3 channel. The averaged rising rates of Ca2+ transients induced by alpha-D-mannose-bovine serum albumin in the head region of Ca(v)2.3-/- sperm were significantly lower than those of Ca(v)2.3+/+ sperm. A computer-assisted sperm motility assay revealed that straight-line velocity and linearity were greater in Ca(v)2.3-/- sperm than those in Ca(v)2.3+/+ sperm. These results suggest that the Ca(v)2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.     

Nitric oxide augments voltage-gated P/Q-type Ca(2+) channels constituting a putative positive feedback loop

P/Q-type Ca(2+) channels, which are postulated to play major roles in synaptic transmission, are regulated in a variety of ways. Ca(2+) currents through P/Q-type Ca(2+) channels (Ca(v)2.1/beta(1a)/alpha(2)delta) heterologously expressed in mammalian cells were recorded using the whole-cell patch clamp method. The oxidant H(2)O(2) increased the current amplitude and the effect was reversed by the reducing agent dithiothreitol (DTT). The stimulatory effect of H(2)O(2) on the Ca(2+) current was mimicked by the NO donors, SNAP, and diethylamine NONOate, and reversed by the reducing agent DTT. The presence of a soluble guanylate cyclase inhibitor did not abolish the ability of SNAP to increase the Ca(2+) current. Adenovirus-mediated overexpression of nitric oxide synthase in combination with application of the Ca(2+) ionophore A23187 also increased the Ca(2+) current amplitude and the effect was again reversed by DTT. The NOS inhibitor L-NAME abolished the stimulatory effect of A23187, and A23187 did not change the Ca(2+) currents in the cells treated with control adenovirus particles. The time course of the decline of the Ca(2+) current, but not of the Ba(2+) current, in response to repeated depolarization was markedly slowed by adenovirus-mediated overexpression of nitric oxide synthase. The results demonstrate that nitric oxide enhances the channel activity by promoting oxidation and suggest that Ca(2+), nitric oxide synthase, and nitric oxide could constitute a positive feedback loop for regulation of voltage-gated P/Q-type Ca(2+) channels.     

Mg(2+) block unmasks Ca(2+)/Ba(2+) selectivity of alpha1G T-type calcium channels

We have examined permeation by Ca(2+) and Ba(2+), and block by Mg(2+), using whole-cell recordings from alpha1G T-type calcium channels stably expressed in HEK 293 cells. Without Mg(o)(2+), inward currents were comparable with Ca(2+) and Ba(2+). Surprisingly, three other results indicate that alpha1G is actually selective for Ca(2+) over Ba(2+). 1) Mg(2+) block is approximately 7-fold more potent with Ba(2+) than with Ca(2+). With near-physiological (1 mM) Mg(o)(2+), inward currents were approximately 3-fold larger with 2 mM Ca(2+) than with 2 mM Ba(2+). The stronger competition between Ca(2+) and Mg(2+) implies that Ca(2+) binds more tightly than Ba(2+). 2) Outward currents (carried by Na(+)) are blocked more strongly by Ca(2+) than by Ba(2+). 3) The reversal potential is more positive with Ca(2+) than with Ba(2+), thus P(Ca) > P(Ba). We conclude that alpha1G can distinguish Ca(2+) from Ba(2+), despite the similar inward currents in the absence of Mg(o)(2+). Our results can be explained by a 2-site, 3-barrier model if Ca(2+) enters the pore 2-fold more easily than Ba(2+) but exits the pore at a 2-fold lower rate.     

Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics

Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q-type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.     

Characterization of Ca(2+) signaling pathways in human mesenchymal stem cells

Human mesenchymal stem cells (HMSC) have the potential to differentiate into many cell types. The physiological properties of HMSCs including their Ca(2+) signaling pathways, however, are not well understood. We investigated Ca(2+) influx and release functions in HMSCs. In Ca(2+) imaging experiments, spontaneous Ca(2+) oscillations were observed in 36 of 50 HMSCs. The Ca(2+) oscillations were completely blocked by the application of 10 micro M cyclopiazonic acid (CPA) or 1 micro M thapsigargin (TG). A brief application of 1 micro M acetylcholine (ACh) induced a transient increase of [Ca(2+)](i) but the application of caffeine (10 mM) did not induce any Ca(2+) transient. When the stores were depleted with Ca(2+)-ATPase blockers (CPA or TG) or muscarinic agonists (ACh), store-operated Ca(2+) (SOC) entry was observed. Using the patch-clamp technique, store-operated Ca(2+) currents (I(SOC)) could be recorded in cells treated with ACh or CPA, but voltage-operated Ca(2+) currents (VOCCs) were not elicited in most of the cells (17/20), but in 15% of cells examined, small dihydropyridine (DHP)-sensitive Ca(2+) currents were recorded. Using RT-PCR, mRNAs were detected for inositol 1,4,5-trisphosphate receptor (InsP(3)R) type I, II, and III and DHP receptors alpha1A and alpha1H were detected, but mRNA was not detected for ryanodine receptor (RyR) or N-type Ca(2+) channels. These results suggest that in undifferentiated HMSCs, Ca(2+) release is mediated by InsP(3)Rs and Ca(2+) entry through plasma membrane is mainly mediated by the SOCs channels with a little contribution of VOCCs.     

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.