Modified sympathetic nerve system activity with overexpression of the voltage-dependent calcium channel beta3 subunit

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory beta3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a beta3-overexpressing transgenic (beta3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of beta3-null and CaV2.2 (alpha1B)-null mice. The beta3-Tg mice showed increased expression of the beta3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The beta3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the beta3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the beta3-Tg mice, whereas beta3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the beta3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of beta3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the beta3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Decreased calcium channel currents and facilitated epinephrine release in the Ca channel β3 subunit-null mice

The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. The β3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in β3-null mice to determine the role of the β3 subunit in catecholamine release from the adrenal chromaffin system.Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The β3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in β3-null mice, while patch-clamp recordings showed a significant reduction in Ca²⁺-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production.In conclusion, our results indicate the importance of the β3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells.     

Regulation of Ca-sensitive inactivation of a 1-type Ca2+ channel by specific domains of beta subunits.

Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.     

Functional properties of cardiac L-type calcium channels transiently expressed in HEK293 cells. Roles of alpha 1 and beta subunits

The cardiac dihydropyridine-sensitive calcium channel was transiently expressed in HEK293 cells by transfecting the rabbit cardiac calcium channel alpha 1 subunit (alpha 1C) alone or in combination with the rabbit calcium channel beta subunit cloned from skeletal muscle. Transfection with alpha 1C alone leads to the expression of inward, voltage-activated, calcium or barium currents that exhibit dihydropyridine sensitivity and voltage- as well as calcium-dependent inactivation. Coexpression of the skeletal muscle beta subunit increases current density and the number of high-affinity dihydropyridine binding sites and also affects the macroscopic kinetics of the current. Recombinant alpha 1C beta channels exhibit a slowing of activation and a faster inactivation rate when either calcium or barium carries the charge. Our data suggest that both an increase in the number of channels as well as modulatory effects on gating underlie the modifications observed upon beta subunit coexpression.     

The neuronal beta 4 subunit increases the unitary conductance of L-type voltage-gated calcium channels in PC12 cells

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.     

Voltage-dependent facilitation of a neuronal alpha 1C L-type calcium channel.

Calcium entry into excitable cells through voltage-gated calcium channels can be influenced by both the rate and pattern of action potentials. We report here that a cloned neuronal alpha 1C L-type calcium channel can be facilitated by positive pre-depolarization. Both calcium and barium were effective as charge carriers in eliciting voltage-dependent facilitation. The induction of facilitation was shown to be independent of intracellular calcium levels, G-protein interaction and the level of phosphatase activity. Facilitation was reduced by the injection of inhibitors of protein kinase A and required the coexpression of a calcium channel beta subunit. In contrast, three neuronal non-L-type calcium channels, alpha 1A, alpha 1B and alpha 1E, were not subject to voltage-dependent facilitation when coexpressed with a beta subunit. The results indicate that the mechanism of neuronal L-type calcium channel facilitation involves the interaction of alpha 1 and beta subunits and is dependent on protein kinase A activity. The selective voltage-dependent modulation of L-type calcium channels is likely to play an important role in neuronal physiology and plasticity.     

Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit

The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Gamma 1 subunit interactions within the skeletal muscle L-type voltage-gated calcium channels

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha(1) and auxiliary alpha(2)delta, beta, and gamma subunits. The gamma subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma is emphasized by epileptic stargazer mice that lack gamma(2). In this study, we examined the molecular basis of interaction between skeletal gamma(1) and the calcium channel. Our data show that the alpha(1)1.1, beta(1a), and alpha(2)delta subunits are still associated in gamma(1) null mice. Reexpression of gamma(1) and gamma(2) showed that gamma(1), but not gamma(2), incorporates into gamma(1) null channels. By using chimeric constructs, we demonstrate that the first half of the gamma(1) subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma(1) null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma(1), we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma(1) and alpha(2)delta are not associated. Moreover, alpha(1)1.1 and gamma(1) subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma(1) and alpha(1)1.1 subunits. Our data demonstrate that the first half of gamma(1) subunit is required for association with the channel through alpha(1)1.1. Because subunit interactions are conserved, these studies have broad implications for gamma heterogeneity, function and subunit association with voltage-gated calcium channels.     

Several structural domains contribute to the regulation of N-type calcium channel inactivation by the beta 3 subunit

Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.     

Calcium channel beta subunit promotes voltage-dependent modulation of alpha 1 B by G beta gamma

Voltage-dependent calcium channels (VDCCs) are heteromultimers composed of a pore-forming alpha1 subunit and auxiliary subunits, including the intracellular beta subunit, which has a strong influence on the channel properties. Voltage-dependent inhibitory modulation of neuronal VDCCs occurs primarily by activation of G-proteins and elevation of the free G beta gamma dimer concentration. Here we have examined the interaction between the regulation of N-type (alpha 1 B) channels by their beta subunits and by G beta gamma dimers, heterologously expressed in COS-7 cells. In contrast to previous studies suggesting antagonism of G protein inhibition by the VDCC beta subunit, we found a significantly larger G beta gamma-dependent inhibition of alpha 1 B channel activation when the VDCC alpha 1 B and beta subunits were coexpressed. In the absence of coexpressed VDCC beta subunit, the G beta gamma dimers, either expressed tonically or elevated via receptor activation, did not produce the expected features of voltage-dependent G protein modulation of N-type channels, including slowed activation and prepulse facilitation, while VDCC beta subunit coexpression restored all of the hallmarks of G beta gamma modulation. These results suggest that the VDCC beta subunit must be present for G beta gamma to induce voltage-dependent modulation of N-type calcium channels.     

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation

It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.     

Involvement of the calcium channel beta3 subunit in olfactory signal transduction

Despite the expression of voltage-dependent Ca2+ channels in nasal turbinate epithelium, their role in odorant chemosensation has remained obscure. Therefore, we investigated olfactory neurotransduction in beta3-deficient mice. RT-PCR and Western blots confirmed the expression of various types of Ca2+ channels in the nasal turbinate. Electrophysiological evaluations revealed that beta3-null mice had a 60% reduction in the high-voltage-dependent Ca2+ currents in olfactory receptor neurons due to reduced N- and L-type channel currents. The beta3-null mice showed increased olfactory neuronal activity to triethylamine, and this effect was mimicked by the perfusion of the specific N-type Ca2+ channel inhibitor omega-conotoxin GVIA in the electro-olfactogram. Diluted male urine odors induced higher Fos immunoreactivity in the main olfactory bulbs of beta3-deficient mice, indicating enhanced signal transduction of odor information in these mice. Our data indicate the involvement of voltage-dependent Ca2+ channels and importance of the beta3 subunit in olfactory signal transduction.     

Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: studies with antibodies against the alpha subunits

Polyclonal antibodies (PAC-2) against the purified skeletal muscle calcium channel were prepared and shown to be directed against alpha subunits of this protein by immunoblotting and immunoprecipitation. These polypeptides have an apparent molecular weight of 162,000 without reduction of disulfide bonds. Under conditions where the functional properties of the purified skeletal muscle calcium channel are retained, beta subunits (Mr 50,000) and gamma subunits (Mr 33,000) are coprecipitated, demonstrating specific noncovalent association of these three polypeptides in the purified skeletal muscle channel. PAC-2 immunoprecipitated cardiac calcium channels labeled with [3H]isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- (methoxycarbonyl)pyridine-3-carboxylate ([3H]PN200-110) at a 3-fold higher concentration than skeletal muscle channels. Preincubation with cardiac calcium channels blocked only 49% of the immunoreactivity of PAC-2 toward skeletal muscle channels, indicating that these two proteins have both homologous and distinct epitopes. The immunoreactive component of the cardiac calcium channel was identified by immunoprecipitation and polyacrylamide gel electrophoresis as a polypeptide with an apparent molecular weight of 170,000 before reduction of disulfide bonds and 141,000 after reduction, in close analogy with the properties of the alpha 2 subunits of the skeletal muscle channel. It is concluded that these two calcium channels have a homologous, but distinct, alpha subunit as a major polypeptide component.     

Ca2+ channel currents and contraction in CaVbeta3-deficient ileum smooth muscle from mouse

Voltage activated L-type Ca(2+) channels are the principal Ca(2+) channels in intestinal smooth muscle cells. They comprise the ion conducting Ca(V)1 pore and the ancillary subunits alpha(2)delta and beta. Of the four Ca(V)beta subunits Ca(V)beta(3) is assumed to be the relevant Ca(V)beta protein in smooth muscle. In protein lysates isolated from mouse ileum longitudinal smooth muscle we could identify the Ca(V)1.2, Ca(V)alpha(2), Ca(V)beta(2) and Ca(V)beta(3) proteins, but not the Ca(V)beta(1) and Ca(V)beta(4) proteins. Protein levels of Ca(V)1.2, Ca(V)alpha(2) and Ca(V)beta(2) are not altered in ileum smooth muscle obtained from Ca(V)beta(3)-deficient mice indicating that there is no compensatory increase of the expression of these channel proteins. Neither the Ca(V)beta(2) nor the other Ca(V)beta proteins appear to substitute for the lacking Ca(V)beta(3). L-type Ca(2+) channel properties including current density, inactivation kinetics as well as Cd(2+)- and dihydropyridine sensitivity were identical in cells of both genotypes suggesting that they do not require the presence of a Ca(V)beta(3) protein. However, a key hallmark of the Ca(V)beta modulation of Ca(2+) current, the hyperpolarisation of channel activation is slightly but significantly reduced by 4 mV. In addition to L-type Ca(2+) currents T-type Ca(2+) currents could be recorded in the murine ileum smooth muscle cells, but T-type currents were not affected by the lack of Ca(V)beta(3). Both proteins, Ca(V)beta(2) and Ca(V)beta(3) are localized near the plasma membrane and the localization of Ca(V)beta(2) is not altered in Ca(V)beta(3) deficient cells. Spontaneous contractions and potassium and carbachol induced contractions are not significantly different between ileum longitudinal smooth muscle strips from mice of both genotypes. In summary the data show that in ileum smooth muscle cells, Ca(V)beta(3) has only subtle effects on L-type Ca(2+) currents, appears not to be required for spontaneous and potassium induced contraction but might have a function beyond being a Ca(2+) channel subunit.     

Voltage and calcium use the same molecular determinants to inactivate calcium channels

During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.     

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.     

Functional roles of the extracellular segments of the sodium channel alpha subunit in voltage-dependent gating and modulation by beta1 subunits.

Voltage-gated sodium channels consist of a pore-forming alpha subunit associated with beta1 subunits and, for brain sodium channels, beta2 subunits. Although much is known about the structure and function of the alpha subunit, there is little information on the functional role of the 16 extracellular loops. To search for potential functional activities of these extracellular segments, chimeras were studied in which an individual extracellular loop of the rat heart (rH1) alpha subunit was substituted for the corresponding segment of the rat brain type IIA (rIIA) alpha subunit. In comparison with rH1, wild-type rIIA alpha subunits are characterized by more positive voltage-dependent activation and inactivation, a more prominent slow gating mode, and a more substantial shift to the fast gating mode upon coexpression of beta1 subunits in Xenopus oocytes. When alpha subunits were expressed alone, chimeras with substitutions from rH1 in five extracellular loops (IIS5-SS1, IISS2-S6, IIIS1-S2, IIISS2-S6, and IVS3-S4) had negatively shifted activation, and chimeras with substitutions in three of these (IISS2-S6, IIIS1-S2, and IVS3-S4) also had negatively shifted steady-state inactivation. rIIA alpha subunit chimeras with substitutions from rH1 in five extracellular loops (IS5-SS1, ISS2-S6, IISS2-S6, IIIS1-S2, and IVS3-S4) favored the fast gating mode. Like wild-type rIIA alpha subunits, all of the chimeric rIIA alpha subunits except chimera IVSS2-S6 were shifted almost entirely to the fast gating mode when coexpressed with beta1 subunits. In contrast, substitution of extracellular loop IVSS2-S6 substantially reduced the effectiveness of beta1 subunits in shifting rIIA alpha subunits to the fast gating mode. Our results show that multiple extracellular loops influence voltage-dependent activation and inactivation and gating mode of sodium channels, whereas segment IVSS2-S6 plays a dominant role in modulation of gating by beta1 subunits. Evidently, several extracellular loops are important determinants of sodium channel gating and modulation.