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.     

Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen

The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.     

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.     

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Coexpression of the rat beta 1 subunit with rat brain and skeletal muscle sodium channel alpha subunits in Xenopus oocytes normalizes currents by accelerating sodium current decay kinetics, shifting steady state availability relationships, and accelerating recovery from inactivation. Unlike brain and skeletal muscle, the heart alpha subunit expressed without beta 1 has native-like decay kinetics in oocytes. Messenger RNA for beta 1 has been found in heart, but whether and how it affects cardiac sodium channel function are unclear. We studied coexpression of human heart alpha subunit with beta 1 in Xenopus oocytes using two microelectrode voltage-clamp and macropatch techniques. Coexpression with beta 1 caused a significant positive shift of 3-7 mV in the midpoint of the steady state inactivation relationship but did not affect single-channel conductance, activation, current decay, or recovery from inactivation. Sensitivity to lidocaine block, however, was decreased for both resting state block (Kd = 0.5-1.3 mM) and phasic block in response to pulse trains, but inactivated state block was not affected (Kd = approximately 10 microM). Coexpression with beta 1 increased the rate of recovery from lidocaine block, which accounted for the major part of the observed differences in tonic and phasic block. A beta 1 construct with the cytoplasmic tail removed also produced these effects, demonstrating that the beta 1 cytoplasmic tail was not involved in altering lidocaine block. We conclude that the beta 1 subunit is capable of affecting function of the cardiac sodium channel in oocytes by decreasing tonic and phasic lidocaine block with small effects on gating.     

Activation of a metabotropic glutamate receptor and protein kinase C reduce the extent of inactivation of the K+ channel Kv1.1/Kvbeta1.1 via dephosphorylation of Kv1.1

Various brain K+ channels, which may normally exist as complexes of alpha (pore-forming) and beta (auxiliary) subunits, were subjected to regulation by metabotropic glutamate receptors. Kv1.1/Kvbeta1.1 is a voltage-dependent K+ channel composed of alpha and beta proteins that are widely expressed in the brain. Expression of this channel in Xenopus oocytes resulted in a current that had fast inactivating and noninactivating components. Previously we showed that basal and protein kinase A-induced phosphorylation of the alpha subunit at Ser-446 decreases the fraction of the noninactivating component. In this study we investigated the effect of protein kinase C (PKC) on the channel. We showed that a PKC-activating phorbol ester (phorbol 12-myristate 13-acetate (PMA)) increased the noninactivating fraction via activation of a PKC subtype that was inhibited by staurosporine and bisindolylmaleimide but not by calphostin C. However, it was not a PKC-induced phosphorylation but rather a dephosphorylation that mediated the effect. PMA reduced the basal phosphorylation of Ser-446 significantly in plasma membrane channels and failed to affect the inactivation of channels having an alpha subunit that was mutated at Ser-446. Also, the activation of coexpressed mGluR1a known to activate phospholipase C mimicked the effect of PMA on the inactivation via induction of dephosphorylation at Ser-446. Thus, this study identified a potential neuronal pathway initiated by activation of metabotropic glutamate receptor 1a coupled to a signaling cascade that possibly utilized PKC to induce dephosphorylation and thereby to decrease the extent of inactivation of a K+ channel.     

Properties of Ba2+ currents arising from human alpha1E and alpha1Ebeta3 constructs expressed in HEK293 cells: physiology, pharmacology, and comparison to native T-type Ba2+ currents

Currents arising from human alpha1E and alpha1Ebeta3 Ca2+ channel subunits expressed in HEK-293 cells were examined with whole-cell recording methods and compared to properties of T-current in DRG neurons studied under identical ionic conditions. Coexpression of alpha1E subunit with the beta3 subunit shifted activation to more negative potentials. Activation and deactivation of both variants were comparable at most voltages, with deactivation becoming faster, but less voltage-dependent, at more negative potentials. The inactivation time course for alpha1E and alpha1Ebeta3 currents was best described by at least two exponential components. Recovery from inactivation was markedly voltage-dependent and similar for both constructs. In comparison to alpha1E and alpha1Ebeta3 constructs, T current activation was shifted to more negative potentials, activation was typically slower, deactivation exhibited a steeper voltage-dependence, and recovery from inactivation was less voltage-dependent. Over most of the activation range, native T current inactivated more completely and in a single exponential fashion. Despite some pharmacological similarities (e.g. octanol, barbiturates) between alpha1E and T-type currents, aspects of blockade by amiloride and phenytoin appear to distinguish alpha1E current from T-type currents. The results define several distinguishing features of alpha1E currents that distinguish them from native T-type currents.     

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.     

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We have expressed the alpha 1 A calcium channel subunit alone, and in combination with different beta subunits, and investigated the effect of the external Ba2+ concentration on the voltage dependence of activation. Increasing the external Ba2+ concentration from 2.5 to 40 mM induced in all cases a depolarising shift of the potential for half-activation. The magnitude of this shift however, was different depending on whether the alpha 1 A subunit was expressed alone or with a beta subunit. Consistently, calculated external surface-charge density and potential were larger when a beta subunit was expressed. These results suggest that expression of an auxiliary subunit can influence calcium channel gating by modifying the sensitivity of the voltage sensor to the membrane potential profile.     

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.     

Multiple modulation pathways of calcium channel activity by a beta subunit. Direct evidence of beta subunit participation in membrane trafficking of the alpha1C subunit

In order to study the precise mechanisms of alpha1 subunit modulation by an auxiliary beta subunit of voltage-dependent calcium channels, a recombinant beta3 subunit fusion protein was produced and introduced into oocytes that express the human alpha1C subunit. Injection of the beta3 subunit protein rapidly modulated the current kinetics and voltage dependence of activation, whereas massive augmentation of peak current amplitude occurred over a longer time scale. Consistent with the latter, a severalfold increase in the amount of the alpha1C subunit in the plasma membrane was detected by quantitative confocal laser-scanning microscopy after beta3 subunit injection. Pretreatment of oocytes with bafilomycin A1, a vacuolar type H+-ATPase inhibitor, abolished the increase of the alpha1C subunit in the plasma membrane, attenuated current increase, but did not affect the modulation of current kinetics and voltage dependence by the beta3 subunit. These results provide clear evidence that the beta subunit modifies the calcium channel complex in a binary fashion; one is an allosteric modulation of the alpha1 subunit function and the other is a chaperoning of the alpha1 subunit to the plasma membrane.     

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.     

The expression of voltage-dependent calcium channel beta subunits in human cerebellum

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.     

G-Protein-dependent facilitation of neuronal alpha1A, alpha1B, and alpha1E Ca channels

Modulation of neuronal voltage-gated Ca channels has important implications for synaptic function. To investigate the mechanisms of Ca channel modulation, we compared the G-protein-dependent facilitation of three neuronal Ca channels. alpha1A, alpha1B, or alpha1E subunits were transiently coexpressed with alpha2-deltab and beta3 subunits in HEK293 cells, and whole-cell currents were recorded. After intracellular dialysis with GTPgammaS, strongly depolarized conditioning pulses facilitated currents mediated by each Ca channel type. The magnitude of facilitation depended on current density, with low-density currents being most strongly facilitated and high-density currents often lacking facilitation. Facilitating depolarizations speeded channel activation approximately 1.7-fold for alpha1A and alpha1B and increased current amplitudes by the same proportion, demonstrating equivalent facilitation of G-protein-inhibited alpha1A and alpha1B channels. Inactivation typically obscured facilitation of alpha1E current amplitudes, but the activation kinetics of alpha1E currents showed consistent and pronounced G-protein-dependent facilitation. The onset and decay of facilitation had the same kinetics for alpha1A, alpha1B, and alpha1E, suggesting that Gbeta gamma dimers dissociate from and reassociate with these Ca channels at very similar rates. To investigate the structural basis for N-type Ca channel modulation, we expressed a mutant of alpha1B missing large segments of the II-III loop and C terminus. This deletion mutant exhibited undiminished G-protein-dependent facilitation, demonstrating that a Gbeta gamma interaction site recently identified within the C terminus of alpha1E is not required for modulation of alpha1B.     

Dopaminergic modulation of sodium current in hippocampal neurons via cAMP-dependent phosphorylation of specific sites in the sodium channel alpha subunit

Phosphorylation of brain Na+ channel alpha subunits by cAMP-dependent protein kinase (PKA) decreases peak Na+ current in cultured brain neurons and in mammalian cells and Xenopus oocytes expressing cloned brain Na+ channels. We have studied PKA regulation of Na+ channel function by activation of D1-like dopamine receptors in acutely isolated hippocampal neurons using whole-cell voltage-clamp recording techniques. The D1 agonist SKF 81297 reversibly reduced peak Na+ current in a concentration-dependent manner. No changes in the voltage dependence or kinetics of activation or inactivation were observed. This effect was mediated by PKA, as it was mimicked by application of the PKA activator Sp-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate(cBIMPS) and was inhibited by the specific PKA inhibitor peptide PKAI5-24. cBIMPS had similar effects on type IIA brain Na+ channel alpha subunits expressed in tsA-201 cells, but no effect was observed on a mutant Na+ channel alpha subunit in which serine residues in five PKA phosphorylation sites in the intracellular loop connecting domains I and II (LI-II) had been replaced by alanine. A single mutation, S573A, similarly eliminated cBIMPS modulation. Thus, activation of D1-like dopamine receptors results in PKA-dependent phosphorylation of specific sites in LI-II of the Na+ channel alpha subunit, causing a reduction in Na+ current. Such modulation is expected to exert a profound influence on overall neuronal excitability. Dopaminergic input to the hippocampus from the mesocorticolimbic system may exert this influence in vivo.     

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.     

Tryptophan synthase mutations that alter cofactor chemistry lead to mechanism-based inactivation

Mutations in the pyridoxal phosphate binding site of the tryptophan synthase beta subunit (S377D and S377E) alter cofactor chemistry [Jhee, K.-H., et al. (1998) J. Biol. Chem. 273, 11417-11422]. We now report that the S377D, S377E, and S377A beta2 subunits form alpha2 beta2 complexes with the alpha subunit and activate the alpha subunit-catalyzed cleavage of indole 3-glycerol phosphate. The apparent Kd for dissociation of the alpha and beta subunits is unaffected by the S377A mutation but is increased up to 500-fold by the S377D and S377E mutations. Although the three mutant alpha2 beta2 complexes exhibit very low activities in beta elimination and beta replacement reactions catalyzed at the beta site in the presence of Na+, the activities and spectroscopic properties of the S377A alpha2 beta2 complex are partially repaired by addition of Cs+. The S377D and S377E alpha2 beta2 complexes, unlike the wild-type and S377A alpha2 beta2 complexes and the mutant beta2 subunits, undergo irreversible substrate-induced inactivation by L-serine or by beta-chloro-L-alanine. The rates of inactivation (kinact) are similar to the rates of catalysis (kcat). The partition ratios are very low (kcat/kinact = 0.25-3) and are affected by alpha subunit ligands and monovalent cations. The inactivation product released by alkali was shown by HPLC and by fluorescence, absorption, and mass spectroscopy to be identical to a compound previously synthesized from pyridoxal phosphate and pyruvate. We suggest that alterations in the cofactor chemistry that result from the engineered Asp377 in the active site of the beta subunit may promote the mechanism-based inactivation.     

G-protein beta-subunit specificity in the fast membrane-delimited inhibition of Ca2+ channels

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.     

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The activity of the major intracellular protein phosphatase, protein phosphatase 2A (PP2A), is determined by the nature of the associated regulatory subunit. A new family of human PP2A regulatory subunits has recently been identified. Three of these subunits, B56beta, B56delta, and B56epsilon, are most highly expressed in brain, while the B56alpha and B56gamma isoforms are highly expressed in cardiac and skeletal muscle. Genes PPP2R5A-PPP2R5E encoding the phosphatase regulatory proteins B56alpha, B56beta, B56gamma, B56delta, and B56epsilon have now been mapped by fluorescence in situ hybridization to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12, respectively.