Alpha2delta Subunits Research Articles

Effects of mutations causing hypokalaemic periodic paralysis on the skeletal muscle L-type Ca2+ channel expressed in Xenopus laevis oocytes.

1. A truncated form of the rabbit alpha1S Ca2+ channel subunit (alpha1SDeltaC) was expressed with the beta1b, alpha2delta and gamma auxiliary subunits in Xenopus laevis oocytes. After 5-7 days, skeletal muscle L-type currents were measured (469 +/- 48 nA in 10 mM Ba2+). All three of the auxiliary subunits were necessary to record significant L-type current. A rapidly inactivating, dihydropyridine-insensitive endogenous Ba2+ current was observed in oocytes expressing the auxiliary subunits without an exogenous alpha subunit. Expression of full-length alpha1S gave 10-fold smaller currents than the truncated form. 2. Three missense mutations causing hypokalaemic periodic paralysis (R528H in domain II S4 of the alpha1S subunit; R1239H and R1239G in domain IV S4) were introduced into alpha1SDeltaC and expressed in oocytes. L-type current was separated from the endogenous current by nimodipine subtraction. All three of the mutations reduced L-type current amplitude ( approximately 40 % for R528H, approximately 60-70 % for R1239H and R1239G). 3. The disease mutations altered the activation properties of L-type current. R528H shifted the G(V) curve approximately 5 mV to the left and modestly reduced the voltage dependence of the activation time constant, tauact. R1239H and R1239G shifted the G(V) curve approximately 5-10 mV to the right and dramatically slowed tauact at depolarized test potentials. 4. The voltage dependence of steady-state inactivation was not significantly altered by any of the disease mutations. 5. Wild-type and mutant L-type currents were also measured in the presence of (-)-Bay K8644, which boosted the amplitude approximately 5- to 7-fold. The effects of the mutations on the position of the G(V) curve and the voltage dependence of tauact were essentially the same as in the absence of agonist. Bay K-enhanced tail currents were slowed by R528H and accelerated by R1239H and R1239G. 6. We conclude that the domain IV mutations R1239H and R1239G have similar effects on the gating properties of the skeletal muscle L-type Ca2+ channel expressed in Xenopus oocytes, while the domain II mutation R528H has distinct effects. This result implies that the location of the substitutions is more important than their degree of conservation in determining their biophysical consequences.

The effect of alpha2-delta and other accessory subunits on expression and properties of the calcium channel alpha1G.

1. The effect has been examined of the accessory alpha2-delta and beta subunits on the properties of alpha1G currents expressed in monkey COS-7 cells and Xenopus oocytes. 2. In immunocytochemical experiments, the co-expression of alpha2-delta increased plasma membrane localization of expressed alpha1G and conversely, the heterologous expression of alpha1G increased immunostaining for endogenous alpha2-delta, suggesting an interaction between the two subunits. 3. Heterologous expression of alpha2-delta together with alpha1G in COS-7 cells increased the amplitude of expressed alpha1G currents by about 2-fold. This finding was confirmed in the Xenopus oocyte expression system. The truncated delta construct did not increase alpha1G current amplitude, or increase its plasma membrane expression. This indicates that it is the exofacial alpha2 domain that is involved in the enhancement by alpha2-delta. 4. Beta1b also produced an increase of functional expression of alpha1G, either in the absence or the presence of heterologously expressed alpha2-delta, whereas the other beta subunits had much smaller effects. 5. None of the accessory subunits had any marked influence on the voltage dependence or kinetics of the expressed alpha1G currents. These results therefore suggest that alpha2-delta and beta1b interact with alpha1G to increase trafficking of, or stabilize, functional alpha1G channels expressed at the plasma membrane.

On the prosthetic groups of the NiFe sulfhydrogenase from Pyrococcus furiosus: topology, structure, and temperature-dependent redox chemistry.

The sulfhydrogenase complex of Pyrococcus furiosus is an alpha beta gamma delta heterotetramer with both hydrogenase activity (borne by the alpha delta subunits) and sulfur reductase activity (carried by the beta gamma subunits). The beta-subunit contains at least two [4Fe-4S] cubanes and the gamma-subunit contains one [2Fe-2S] cluster and one FAD molecule. The delta-subunit contains three [4Fe-4S] cubanes and the alpha-subunit carries the NiFe dinuclear center. Only three Fe/S signals are observed in EPR-monitored reduction by dithionite, NADPH, or internal substrate upon heating. All other clusters presumably have reduction potentials well below that of the H+/H2 couple. Heat-induced reduction by internal substrate allows, for the first time, EPR monitoring of the NiFe center in a hyperthermophilic hydrogenase, which passes through a number of states, some of which are similar to states previously defined for mesophilic hydrogenases. The complexity of the observed transitions reflects a combination of temperature-dependent activation and temperature-dependent reduction potentials.

Absence of modulation of the expressed calcium channel alpha1G subunit by alpha2delta subunits.

1. The modulatory action of the alpha2delta subunit on various high-voltage-activated calcium channels has been demonstrated previously. However, very little is known about auxiliary subunit modulation of low-voltage-activated (LVA) calcium channels. We have examined the modulation of the alpha1G subunit corresponding to the neuronal T-type calcium channel by the ubiquitously expressed alpha2delta-1 and brain-specific alpha2delta-3 subunits. 2. The alpha1G subunit was expressed alone or in combination with either the alpha2delta-1 or alpha2delta-3 subunit in human embryonic kidney (HEK 293) cells and whole-cell barium currents were measured. The current density-voltage relationships for peak and sustained current, kinetics of current activation and inactivation, voltage dependence of current inactivation and time course of the recovery from inactivation were analysed for each type of expressed channel. No significant difference was found for any of the examined parameters. 3. These results suggest that the LVA alpha1G channel is not regulated by known auxiliary alpha2delta subunits.

Subunit Interface Selectivity of the α-Neurotoxins for the Nicotinic Acetylcholine Receptor

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.

Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line.

1. Voltage-dependent calcium channels (VDCCs) show a highly non-uniform distribution in many cell types, including neurons and other polarized secretory cells. We have examined whether this can be mimicked in a polarized epithelial cell line (Madin-Darby canine kidney), which has been used extensively to study the targeting of proteins. 2. We expressed the VDCC alpha1A, alpha1B or alpha1C subunits either alone or in combination with accessory subunits alpha2-delta and the different beta subunits, and examined their localization immunocytochemically. An alpha1 subunit was only targeted to the plasma membrane if co-expressed with the accessory subunits. 3. The combination alpha1C/alpha2-delta and all beta subunits was always localized predominantly to the basolateral membrane. It has been suggested that this is equivalent to somatodendritic targeting in neurons. 4. In contrast, the alpha1B subunit was expressed at the apical membrane with all the accessory subunit combinations, by 24 h after microinjection. This membrane destination shows some parallels with axonal targeting in neurons. 5. The alpha1A subunit was consistently observed at the apical membrane in the combinations alpha1A/alpha2-delta/beta1b or beta4. In contrast, when co-expressed with alpha2-delta/beta2a, alpha1A was clearly targeted to the basolateral membrane. 6. In conclusion, the VDCC alpha1 subunit appears to be the primary determinant for targeting the VDCC complex, but the beta subunit can modify this destination, particularly for alpha1A.

Complexes of the α1C and β 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 Deletion Mutagenesis of the α2δ Calcium Channel Subunit from Porcine Cerebral Cortex: EXPRESSION OF A SOLUBLE FORM OF THE PROTEIN THAT RETAINS [3H]GABAPENTIN BINDING ACTIVITY

The anti-epileptic, anti-hyperalgesic, and anxiolytic agent gabapentin (1-(aminomethyl)-cyclohexane acetic acid or Neurontin) has previously been shown to bind with high affinity to the alpha2delta subunit of voltage-dependent calcium channels (Gee, N. S. , Brown, J. P., Dissanayake, V. U. K., Offord, J., Thurlow, R., and Woodruff, G.N. (1996) J. Biol. Chem. 271, 5768-5776). We report here the cloning, sequencing, and deletion mutagenesis of the alpha2delta subunit from porcine brain. The deduced protein sequence has a 95.9 and 98.2% identity to the rat and human neuronal alpha2 delta sequences, respectively. [3H]Gabapentin binds with a KD of 37.5 +/- 10.4 nM to membranes prepared from COS-7 cells transfected with wild-type porcine alpha2 delta cDNA. Six deletion mutants (B-G) that lack the delta polypeptide, together with varying amounts of the alpha2 component, failed to bind [3H]gabapentin. C-terminal deletion mutagenesis of the delta polypeptide identified a segment (residues 960-994) required for correct assembly of the [3H]gabapentin binding pocket. Mutant L, which lacks the putative membrane anchor in the delta sequence, was found in both membrane-associated and soluble secreted forms. The soluble form was not proteolytically cleaved into separate alpha2 and delta chains but still retained a high affinity (KD = 30.7 +/- 8.1 nM) for [3H]gabapentin. The production of a soluble alpha2delta mutant supports the single transmembrane model of the alpha2 delta subunit and is an important step toward the large-scale recombinant expression of the protein.

The effect of overexpression of auxiliary Ca2+ channel subunits on native Ca2+ channel currents in undifferentiated mammalian NG108-15 cells.

1. High voltage activated (HVA) Ca2+ channels are composed of a pore-forming alpha 1 subunit and the accessory beta and alpha2-delta subunits. However, the subunit composition of low voltage activated (LVA), or T-type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits. 2. Endogenous alpha 1A, B, C, C, and E, and low levels of beta and alpha 2-delta subunit protein were demonstrated in undifferentiated NG108-15 cells. 3. The alpha 2-delta, beta 2a or beta 1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of alpha 2-delta but also of beta 2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above -30 mV, and alpha 2-delta additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T-type currents. In contrast, beta 1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells. 4 These results suggest that in NG108-15 cells, overexpression of the Ca2+ channel accessory subunits alpha 2-delta and beta 2a induce a sustained component of HVA current, and alpha 2-delta also influences the voltage dependence of activation of the LVA current. It is possible that native T-type alpha 1 subunits are not associated with beta subunits.

Role of domain I of neuronal Ca2+ channel alpha1 subunits in G protein modulation.

1. We studied the G protein inhibition of heteromultimeric neuronal Ca2+ channels by constructing a series of chimeric channels between the strongly modulated alpha1B subunit and the alpha1E(rbEII) subunit, which showed no modulation. 2. In parallel studies, alpha1 subunit constructs were co-expressed together with the accessory Ca2+ channel alpha2-delta and beta2a subunits in mammalian (COS-7) cells and Xenopus oocytes. G protein inhibition of expressed Ca2+ channel currents was induced by co-transfection of Gbeta1 and Ggamma2 subunits in COS-7 cells or activation of co-expressed dopamine (D2) receptors by quinpirole (100 nM) in oocytes. 3. The data indicate that transfer of the alpha1B region containing the N-terminal, domain I and the I-II loop (i.e. the alpha1B1-483 sequence), conferred G protein modulation on alpha1E(rbEII), both in terms of a slowing of activation kinetics and a reduction in current amplitude. 4. In contrast, the data are not consistent with the I-II loop and/or the C-terminal forming a unique site for G protein modulation.

Distinctions in Agonist and Antagonist Specificity Conferred by Anionic Residues of the Nicotinic Acetylcholine Receptor

Two anionic residues in the nicotinic acetylcholine receptor, Asp-152 in the alpha-subunit and Asp-174 in the gamma-subunit or the corresponding Asp-180 in the delta-subunit, are presumed to reside near the two agonist binding sites at the alphagamma and alphadelta subunit interfaces of the receptor and have been implicated in electrostatic attraction of cationic ligands. Through site-directed mutagenesis and analysis of state changes in the receptor elicited by agonists, we have distinguished the roles of anionic residues in conferring ligand specificity and ligand-induced state changes. alphaAsp-152 affects agonist and antagonist affinity similarly, whereas gammaAsp-174 and deltaAsp-180 primarily affect agonist affinity. Combining charge neutralization on the alpha subunit with that on the gamma and delta subunits shows an additivity in free energy changes for carbamylcholine and d-tubocurarine, suggesting independent contributions of these residues to stabilizing the bound ligands. Since both aromatic and anionic residues stabilize cationic ligands, we substituted tyrosines (Y) for the aspartyl residues. While the substitution, alphaD152Y, reduced the affinities for agonists and antagonists, the gammaD174Y/deltaD180Y mutations reduced the affinity for agonist binding, but surprisingly enhanced the affinity for d-tubocurarine. To ascertain whether selective changes in agonist binding stem from the capacity of agonists to form the desensitized state of the receptor, carbamylcholine binding was measured in the presence of an allosteric inhibitor, proadifen. Mutant nAChRs carrying alphaD152Q or gammaD174N/deltaD180N show similar reductions in dissociation constants for the desensitized compared with activable receptor state and a similar proadifen concentration dependence. Hence, these mutations influence ligand recognition rather than the capacity of the receptor to desensitize. By contrast, the alphaD200Q mutation diminishes the ratio of dissociation constants for two states and requires higher proadifen concentrations to induce desensitization. Thus, the contributions of alphaAsp-152, gamma/deltaAsp-174/180, and alphaAsp-200 in stabilizing ligand binding can be distinguished by the interactions between agonists and allosteric inhibitors.

Facilitation of rabbit alpha1B calcium channels: involvement of endogenous Gbetagamma subunits.

1. The alpha1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gbetagamma subunits. Using transient expression in COS-7 cells of alpha1B together with the accessory subunits alpha2-delta and beta2a, we have examined the role of endogenous Gbetagamma subunits in the tonic modulation of alpha1B, and compared this with modulation by exogenously expressed Gbetagamma subunits. 2. Prepulse facilitation of control alpha1B/alpha2-delta/beta2a currents was always observed. This suggests the existence of tonic modulation of alpha1B subunits. To determine whether endogenous Gbetagamma is involved in the facilitation observed in control conditions, the betaARK1 Gbetagamma-binding domain (amino acids 495-689) was overexpressed, in order to bind free Gbetagamma subunits. The extent of control prepulse-induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPbetaS also reduced the control facilitation. 3. Co-expression of the Gbeta1gamma2 subunit, together with the alpha1B/alpha2-delta/beta2a calcium channel combination, resulted in a marked degree of depolarizing prepulse-reversible inhibition of the whole-cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid-point of the voltage dependence of activation. Activation of endogenous Gbetagamma subunits by dialysis with GTPgammaS produced a smaller degree of prepulse-reversible inhibition. 4. The rate of reinhibition of alpha1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gbeta1gamma2 than for the decay of facilitation in control cells. Furthermore, betaARK1 (495-689) co-expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gbetagamma in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. 5. These findings indicate that, in this system, the voltage-dependent facilitation of alpha1B that is observed under control conditions occurs as a result of endogenous free Gbetagamma binding to alpha1B.

Identification of novel ligands for the gabapentin binding site on the alpha2delta subunit of a calcium channel and their evaluation as anticonvulsant agents.

As part of a program to investigate the structure-activity relationships of Gabapentin (Neurontin), a number of alkylated analogues were synthesized and evaluated in vitro for binding to the Gabapentin binding site located on the alpha2delta subunit of a calcium channel. A number of other bridged and heterocyclic analogues are also reported along with their in vitro data. Two compounds showing higher affinity than Gabapentin were selected for evaluation in an animal model of epilepsy. One of these compounds, cis-(1S,3R)-(1-(aminomethyl)-3-methylcyclohexyl)acetic acid hydrochloride (19), was shown to be effective in this model with a profile similar to that of Gabapentin itself.

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding.

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.

Effects of phenytoin, carbamazepine, and gabapentin on calcium channels in hippocampal granule cells from patients with temporal lobe epilepsy.

The anticonvulsants phenytoin (PHT), carbamazepine (CBZ), and gabapentin (GBP) are commonly used in the treatment of temporal lobe epilepsy. Ca2+ current modulation has been proposed to contribute to the antiepileptic activity of these drugs. The purpose of this study was to determine the effects of these anticonvulsants on voltage-dependent calcium channels in pathologically altered neurons from patients with chronic temporal lobe epilepsy. Acutely isolated human hippocampal granule cells were examined by using the whole-cell configuration of the patch-clamp technique. PHT and CBZ produced a reversible, concentration-dependent inhibition of high-voltage-activated (HVA) Ca2+ currents without affecting voltage-dependent activation. The concentration-response curves of PHT and CBZ indicated maximal inhibition of 35 and 65%, respectively, with half-maximal inhibition being obtained at 89 and 244 microM, respectively. At therapeutic cerebrospinal fluid (CSF) concentrations, HVA currents were not significantly altered by PHT and CBZ. However, PHT but not CBZ showed a reduction of HVA currents of 16% at a therapeutic whole-brain concentration of 80 microM. In contrast to CBZ, PHT produced a small hyperpolarizing shift in the voltage dependence of steady-state inactivation. PHT, 80 microM, shifted the potential of half-maximal inactivation by -3.1 +/- 0.5 mV (p < 0.05). GBP, which was recently found to bind to the alpha2delta subunit of a neuronal Ca2+ channel, showed no modulation of Ca2+ conductances. These results suggest that, in contrast to GBP and CBZ, modulation of postsynaptic Ca2+ channels can contribute to the anticonvulsant action of PHT in human hippocampal granule cells.

Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Gbeta subunit.

We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (alpha1B + beta1b + alpha2delta subunits) by exogenously applied purified G protein betagamma subunits. In the absence of Gbetagamma, barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified Gbetagamma in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of Gbetagamma-dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant tau for the recovery from facilitation was sensitive to exogenous Gbetagamma, so that the inverse of tau linearly depended on the Gbetagamma concentration. Overall, the data are consistent with a model whereby a single Gbetagamma molecule dissociates from the channel during the prepulse, and that reassociation of Gbetagamma with the channel after the prepulse occurs as a bimolecular reaction.

Subunit regulation of the human brain alpha 1E calcium channel.

The alpha1 subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary alpha2delta and beta subunits. alpha1E channels directed with the expression of Ba2+ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of alpha1E with alpha2bdelta shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of alpha1E with beta2a shifted the peak current relationship by -10 mV, and strongly reduced Ba2+ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 +/- 10%, n = 8) of the Ba2+ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain beta2a and the neuronal alpha2bdelta subunits increased by approximately 10-fold whole-cell Ba2+ currents although coinjection with either beta2a or alpha2bdelta alone failed to significantly increase alpha1E peak currents. Coexpression with beta2a and alpha2bdelta yielded Ba2+ currents with inactivation kinetics similar to the beta2a induced currents, indicating that the neuronal alpha2bdelta subunit has little effect on alpha1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four beta subunit genes. The slower inactivation was unique to alpha1E/beta2a currents. Coexpression with beta1a, beta1b, beta3, and beta4, yielded faster-inactivating Ba2+ currents than currents recorded from the alpha1E subunit alone. Furthermore, alpha1E/alpha2bdelta/beta1a; alpha1E/alpha2bdelta/beta1b; alpha1E/alpha2bdelta/beta3; alpha1E/alpha2bdelta/beta4 channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The beta subunit-induced changes in the properties of alpha1E channel were comparable to modulation effects reported for alpha1C and alpha1A channels with beta3 approximately beta1b > beta1a approximately beta4 >> beta2a inducing fastest to slowest rate of whole-cell inactivation.

Nine L-type Amino Acid Residues Confer Full 1,4-Dihydropyridine Sensitivity to the Neuronal Calcium Channel α1ASubunit: ROLE OF L-TYPE MET1188

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel alpha1 subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; alpha1C-a numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1, 4-dihydropyridine-insensitive alpha1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant alpha1A subunits, coexpressed with alpha2delta and beta1a subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant alpha1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (alpha1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into alpha1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1, 4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1, 4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.

Functional Expression and Characterization of Skeletal Muscle Dihydropyridine Receptors in Xenopus Oocytes

Dihydropyridine receptors in vertebrate skeletal muscle serve a dual role: as voltage sensors for excitation-contraction coupling and as voltage-activated calcium channels. Although they were the first of six classes of calcium channels to be cloned, skeletal muscle dihydropyridine receptors remain the only ones not functionally expressed as calcium channels in Xenopus oocytes, leading to the hypothesis that an interacting component is missing. Using beta1b, an isoform previously found in brain, we have for the first time reconstituted skeletal muscle calcium channel function in Xenopus oocytes. We show that this beta subunit is necessary for functional expression and that the alpha2delta subunit significantly enhances the expressed current. The majority of the alpha1 subunit in skeletal muscle is a truncated form. Here we show that both the full-length and truncated forms produce functional calcium channels in Xenopus oocytes, but the truncated form gives significantly larger currents. In addition, we show that the beta1b transcript is expressed in rat skeletal muscle, although at a much lower level than the abundant beta1a isoform.

Functional expression of rat brain cloned alpha1E calcium channels in COS-7 cells.

The properties of the rat brain alpha1E Ca2+ channel subunit and its modulation by accessory rat brain alpha2-delta and beta1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether alpha1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. alpha1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric alpha1E/alpha2-delta/beta1b channels were largely dictated by the presence of the beta1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. alpha1E/alpha2-delta/beta1b channels did not appear to be regulated by Ca2+-induced inactivation. alpha1E was shown to exhibit a unique pharmacological profile. omega-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst omega-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 micro;M) produced an inhibition of 51 +/- 7%, whereas the DHP agonist S-(-)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the alpha1E Ca2+ channel subunit; we propose that rat brain alpha1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.