cAMP-dependent phosphorylation of the cardiac L-type Ca channel: a missing link?

Cardiac inotropic effects of beta adrenergic agonists occur mainly through an increase in L-type (class C) calcium channel activity. This response has been attributed to phosphorylation of the L-type Ca channel, or a closely associated protein, by the cAMP-dependent protein kinase A (PKA). Among the three subunits forming the cardiac L-type Ca channel (alpha 1, beta and alpha 2-delta), biochemical studies have revealed that two subunits, alpha 1 and beta, are phosphorylated in vitro by protein kinase A, the alpha 1 subunit being the primary target. However, attempts to reconstitute the cAMP-dependent regulation of the expressed class C Ca channel, either in Xenopus oocytes or in cell lines, have provided contradictory results. We were unable to detect cAMP-dependent modulation of class C alpha 1 subunit Ca channels expressed in Xenopus oocytes, even when coinjected with auxiliary subunits beta and alpha 2-delta. Nevertheless, activity of Ca channels recorded from cardiac-mRNA injected oocytes was potentiated by injection of cAMP or PKA, even when expression of the beta subunit was suppressed using antisense oligonucleotide. Taken together, these results indicate that cAMP-dependent regulation does not exclusively involve the alpha 1 and the beta subunits of the Ca channel and suggest that unidentified protein(s), expressed in cardiac tissue, are most likely necessary.     

Protein kinase C-mediated regulation of L-type Ca channels from skeletal muscle requires phosphorylation of the alpha 1 subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the alpha 1 and beta subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the alpha 1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the beta subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the alpha 1 subunit.     

Molecular determinants of calcium-dependent inactivation in cardiac L-type calcium channels

We investigated the nature and structural requirements for Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channel. Investigation of subunit requirements indicates that the interaction of alpha 1 subunit with ancillary subunits, especially beta subunit, is important for this property. Replacement of the putative cytoplasmic regions of the cardiac alpha 1 subunit with skeletal muscle counterparts eliminates Ca(2+)-dependent inactivation, indicating that the site regulated by Ca2+ resides in the cytoplasmic region of the alpha 1 subunit. Deletion of the carboxy-terminal region of the cardiac alpha 1 subunit does not eliminate this property, suggesting that the modulation by protein kinase A may not be involved in this mechanism. Single amino acid substitution that strongly reduces Ca2+ selectivity of Ca2+ channels also eliminates Ca(2+)-dependent inactivation, suggesting the close link between the ion selectivity and Ca(2+)-dependent inactivation.     

Subunit stoichiometry of the epithelial sodium channel

The epithelial Na+ Channel (ENaC) mediates Na+ reabsorption in a variety of epithelial tissues. ENaC is composed of three homologous subunits, termed alpha, beta, and gamma. All three subunits participate in channel formation as the absence of any one subunit results in a significant reduction or complete abrogation of Na+ current expression in Xenopus oocytes. To determine the subunit stoichiometry, a biophysical assay was employed utilizing mutant subunits that display significant differences in sensitivity to channel blockers from the wild type channel. Our results indicate that ENaC is a tetrameric channel with an alpha2 beta gamma stoichiometry, similar to that reported for other cation selective channels, such as Kv, Kir, as well as voltage-gated Na+ and Ca2+ channels that have 4-fold internal symmetry.     

Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.     

Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.     

Structures and functions of calcium channel beta subunits

Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.     

Mechanism of auxiliary subunit modulation of neuronal alpha1E calcium channels

Voltage-gated calcium channels are composed of a main pore-forming alpha1 moiety, and one or more auxiliary subunits (beta, alpha2 delta) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of alpha1E calcium channels in combination with a beta (beta1-beta4) and/or the alpha2 delta subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting alpha2delta with beta subunits, of transfecting two different beta subunits simultaneously, and of COOH-terminal truncation of alpha1E to remove a second beta binding site. The main results are as follows. (a) The alpha2delta and beta subunits modulate alpha1E in fundamentally different ways. The sole effect of alpha2 delta is to increase current density by elevating channel density. By contrast, though beta subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different beta isoforms produce nearly indistinguishable effects on activation. However, beta subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different beta subunits. (d) The modulatory features found here for alpha1E do not generalize uniformly to other alpha1 channel types, as alpha1C activation gating shows marked beta isoform dependence that is absent for alpha1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha1E calcium channels.     

Investigation of the relationship between farrowing environment, sex steroid concentrations and maternal aggression in gilts

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.     

Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels

We report several unexpected findings that provide novel insights into the properties and interactions of the alpha 1 and beta subunits of dihydropyridine-sensitive L-type channels. First, the beta 2a subunit was expressed as multiple species of 68-72 kDa; the 70-72-kDa species arose from post-translational modification. Second, cell fractionation and immunocytochemical studies indicated that the hydrophilic beta 2a subunit, when expressed alone, was membrane-localized. Third, the beta 2a subunit increased the membrane localization of the alpha 1 subunit and the number of cells expressing L-type Ca2+ currents, without affecting the total amount of the expressed alpha 1C subunit. Expression of maximal currents in alpha 1C/beta 2a cotransfected cells paralleled the time course of expression of the beta subunit. Taken together, these results suggest that the beta subunit plays multiple roles in the formation, stabilization, targeting, and modulation of L-type channels.     

Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination

Interactions of G-protein alpha (Galpha) and beta gamma subunits (Gbeta gamma) with N- (alpha1B) and P/Q-type (alpha1A) Ca2+ channels were investigated using the Xenopus oocyte expression system. Gi3alpha was found to inhibit both N- and P/Q-type channels by receptor agonists, whereas Gbeta1 gamma2 was responsible for prepulse facilitation of N-type channels. L-type channels (alpha1C) were not regulated by Galpha or Gbeta gamma. For N-type, prepulse facilitation mediated via Gbeta gamma was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the alpha1C loop 1. Galpha-mediated inhibitions were also impaired by substitution of the alpha1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished Galpha-mediated inhibition. Moreover, a chimera of L-type with the alpha1B loop 1 gained Gbeta gamma-dependent facilitation, whereas an L-type chimera with the N- or P/Q-type C terminus gained Galpha-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for Gbeta gamma and the C termini of P/Q- and N-types for Galpha.     

Ca2+ channel beta3 subunit enhances voltage-dependent relief of G-protein inhibition induced by muscarinic receptor activation and Gbetagamma

The Ca2+ channel beta subunit has been shown to reduce the magnitude of G-protein inhibition of Ca2+ channels. However, neither the specificity of this action to different forms of G-protein inhibition nor the mechanism underlying this reduction in response is known. We have reported previously that coexpression of the Ca2+ channel beta3 subunit causes M2 muscarinic receptor-mediated inhibition of alpha1B Ca2+ currents to become more voltage-dependent. We report here that the beta3 subunit increases the rate of relief of inhibition produced by a depolarizing prepulse and also shifts the voltage dependency of this relief to more hyperpolarized voltages; these effects are likely to be responsible for the reduction of inhibitory response of alpha1B channels to G-protein-mediated inhibition seen after coexpression of the Ca2+ channel beta3 subunit. Additionally, the beta3 subunit alters the rate and voltage dependency of relief of the inhibition produced by coexpressed Gbeta1gamma1, in a manner similar to the changes it produces in relief of M2 receptor-induced inhibition. We conclude that the Ca2+ channel beta3 subunit reduces the magnitude of G-protein inhibition of alpha1B Ca2+ channels by enhancing the rate of dissociation of the G-protein betagamma subunit from the Ca2+ channel alpha1B subunit.     

beta-Fructosidases: a new superfamily of glycosyl-hydrolases

Dihydropyridines (DHPs) block L-type Ca2+ channels more potently at depolarized membrane potentials, consistent with high affinity binding to the inactivated state. Nisoldipine (a DHP antagonist) blocks the smooth muscle channel more potently than the cardiac one, a phenomenon observed not only in native channels but also in expressed channels. We examined whether this tissue specificity was attributable to differences of inactivation in the two channel types. We expressed cardiac or smooth muscle alpha1C subunits in combination with beta2a and alpha2/delta subunits in human embryonic kidney cells, and used 2 mM Ca2+ as the permeant ion. This system thus reproduces the in vivo topology and charge carrier of the channels while facilitating comparison of the two alpha1C splice variants. Both voltage-dependent and isoform-specific sensitivity of 10 nM nisoldipine inhibition of the channel were demonstrated, with the use of -100 mV as the holding potential for fully reprimed channels and -65 mV to populate the inactivated state. Under drug-free conditions, we characterized fast inactivation (1-sec prepulses) and slow inactivation (3 min prepulses) in the two isoforms. Inactivation parameters were not statistically different in the two channel isoforms; if anything, cardiac channels tended to inactivate more than the smooth muscle channels at relevant voltages. Likewise, the voltage-dependent activation was identical in the two isoforms. We thus conclude that the more potent nisoldipine inhibition of smooth muscle versus cardiac L-type Ca2+ channels is not attributable to differences in channel inactivation or activation. Intrinsic, gating-independent DHP receptor binding affinity differences must be invoked to explain the isoform-specific sensitivity of the DHP block.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

The possible function of stone ramparts at the nest entrance of the blackstart

The origin of Ibetanull, the Ca2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) beta1a subunit, was investigated. The density of Ibetanull was similar to that of Idys, the Ca2+ current of myotubes from dysgenic mice lacking the skeletal DHPR alpha1S subunit (-0.6 +/- 0.1 and -0.7 +/- 0.1 pA/pF, respectively). However, Ibetanull activated at significantly more positive potentials. The midpoints of the GCa-V curves were 16.3 +/- 1.1 mV and 11.7 +/- 1.0 mV for Ibetanull and Idys, respectively. Ibetanull activated significantly more slowly than Idys. At +30 mV, the activation time constant for Ibetanull was 26 +/- 3 ms, and that for Idys was 7 +/- 1 ms. The unitary current of normal L-type and beta1-null Ca2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca2+ was 22 +/- 4 fA and 43 +/- 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca2+ channels, which was 84 +/- 9 fA under the same conditions. Ibetanull and Idys have different gating and permeation characteristics, suggesting that the bulk of the DHPR alpha1 subunits underlying these currents are different. Ibetanull is suggested to originate primarily from Ca2+ channels with a DHPR alpha1S subunit. Dysgenic Ca2+ channels may be a minor component of this current. The expression of DHPR alpha1S in beta1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.     

Interaction of the delta and b subunits contributes to F1 and F0 interaction in the Escherichia coli F1F0-ATPase

Interactions of the F1F0-ATPase subunits between the cytoplasmic domain of the b subunit (residues 26-156, bcyt) and other membrane peripheral subunits including alpha, beta, gamma, delta, epsilon, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of bcyt fused to the activation domain of the yeast GAL-4, and delta subunit fused to the DNA binding domain resulted in the strong expression of the beta-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of bcyt fused to glutathione S-transferase (GST) together with the delta subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-bcyt fusion alone had no such effect, indicating that GST-bcyt was protected by the co-expressed delta subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the delta subunit in the cell. The affinity purified GST-bcyt did not contain significant amounts of delta, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified bcyt together with alpha, beta, gamma, delta, and epsilon, or with the same combination except epsilon. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and delta was stabilized by F1 subunits other than epsilon and also suggested that b-delta interaction was important for F1-F0 interaction.