The reconstituted alpha 3 beta 3 delta complex of the thermostable F1-ATPase

Previously we reported that ATPase activity was recovered when the subunit alpha + beta + gamma or alpha + beta + delta of the F1-ATPase from the thermophilic bacterium PS3 were combined under appropriate conditions. Unlike that of holoenzyme (TF1) and the alpha + beta + gamma mixture, ATPase activity of the alpha + beta + delta mixture was heat labile and insensitive to azide inhibition (Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1977) J. Biol. Chem. 252, 3480-3485). Here, the properties of purified subunit complexes were compared in detail with those of native TF1. The subunit stoichiometries of the complexes were determined to be alpha 3 beta 3 gamma 1 and alpha 3 beta 3 delta 1. In general, the properties of the alpha 3 beta 3 gamma complex are very similar to those of TF1, whereas those of the alpha 3 beta 3 delta complex are significantly different. ATPase activity of the alpha 3 beta 3 delta complex is cold labile. The alpha 3 beta 3 delta complex showed a less stringent specificity for substrate and divalent cation than TF1 and the alpha 3 beta 3 gamma complex. Two Km values for ATP were exhibited by the alpha 3 beta 3 delta complex with the lower one being in the range of 0.1 microM. Equilibrium dialysis experiments revealed that the alpha 3 beta 3 delta complex cannot specifically bind ADP in the absence of Mg2+, while TF1 and the alpha 3 beta 3 gamma complex bind about 1 and 3 mol of ADP/mol of enzyme, respectively. ADP-dependent inactivation of the alpha 3 beta 3 delta complex by dicyclohexylcarbodiimide was not observed. The alpha 3 beta 3 gamma complex was readily formed when the gamma subunit was added to the alpha 3 beta 3 delta complex, suggesting that the alpha 3 beta 3 delta complex is not a "dead-end" complex. The cause of thermolability of the alpha 3 beta 3 delta complex appears to be the low stability of the complex itself at high temperature and not due to an unusually low thermostability of the delta subunit.     

Iodo-alpha-conotoxin MI selectively binds the alpha/delta subunit interface of muscle nicotinic acetylcholine receptors

The embryonic mouse muscle nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel formed by alpha1, beta1, delta, and gamma subunits. The receptor contains two ligand binding sites at alpha/delta and alpha/gamma subunit interfaces. [(3)H]Curare preferentially binds the alpha/gamma interface. We describe the synthesis and properties of a high-affinity iodinated ligand that selectively binds the alpha/delta interface. An analogue of alpha-conotoxin MI was synthesized with an iodine attached to Tyr-12 (iodo-alpha-MI). The analogue potently blocks the fetal mouse muscle subtype of nAChR expressed in Xenopus oocytes. It failed, however, to block alpha3beta4, alpha4beta2, or alpha7 nAChRs. Iodo-alpha-MI potently blocks the alpha1beta1delta but not the alpha1beta1gamma subunit combination expressed in Xenopus oocytes indicating selectivity for the alpha/delta subunit interface. Alpha-conotoxin MI was subsequently radioiodinated, and its properties were further evaluated. Saturation experiments indicate that radioiodinated alpha-conotoxin MI binds to TE671 cell homogenates with a Hill slope of 0.95 +/- 0.0094. Kinetic studies indicate that the binding of [(125)I]alpha-conotoxin MI is reversible (k(off) = 0.084 +/- 0.0045 min(-1)); k(on) is 8.5 x 10(7) min(-1) M(-1). The calculated k(d) is 0.98 nM. This potency is approximately 20-fold higher than the unmodified alpha-MI peptide. Unlike [(125)I]alpha-bungarotoxin, [(125)I]alpha-conotoxin MI binding to TE671 cell homogenates is fully displaceable by the small molecule antagonist d-tubocurarine.     

Isolation,structure, and activity of GID,a novel alpha 4/7-conotoxin with an extended N-terminal sequence

Using assay-directed fractionation of Conus geographus crude venom, we isolated alpha-conotoxin GID, which acts selectively at neuronal nicotinic acetylcholine receptors (nAChRs). Unlike other neuronally selective alpha-conotoxins, alpha-GID has a four amino acid N-terminal tail, gamma-carboxyglutamate (Gla), and hydroxyproline (O) residues, and lacks an amidated C terminus. GID inhibits alpha 7 and alpha 3 beta 2 nAChRs with IC(50) values of 5 and 3 nm, respectively and is at least 1000-fold less potent at the alpha 1 beta 1 gamma delta, alpha 3 beta 4, and alpha 4 beta 4 combinations. GID also potently inhibits the alpha 4 beta 2 subtype (IC(50) of 150 nm). Deletion of the N-terminal sequence (GID Delta 1-4) significantly decreased activity at the alpha 4 beta 2 nAChR but hardly affected potency at alpha 3 beta 2 and alpha 7 nAChRs, despite enhancing the off-rates at these receptors. In contrast, Arg(12) contributed to alpha 4 beta 2 and alpha 7 activity but not to alpha 3 beta 2 activity. The three-dimensional structure of GID is well defined over residues 4-19 with a similar motif to other alpha-conotoxins. However, despite its influence on activity, the tail appears to be disordered in solution. Comparison of GID with other alpha 4/7-conotoxins which possess an NN(P/O) motif in loop II, revealed a correlation between increasing length of the aliphatic side-chain in position 10 (equivalent to 13 in GID) and greater alpha 7 versus alpha 3 beta 2 selectivity.     

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.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.     

Reconstitution of adenosine triphosphatase of thermophilic bacterium from purified individual subunits.

1. Five subunits (alpha, beta, gamma, delta, and epsilon) of an ATPase from a thermophilic bacterium PS3 were purified in the presence of 8 M urea by ion exchange chromatography. Then the ATPase activity was reconstituted by mixing the subunit solutions and incubating them at 20-45 degrees, at pH 6.3 to 7.0. 2. Mixtures containing beta + gamma or alpha + beta + delta regained ATP-hydrolyzing activity, but mixtures of alpha + beta and beta + delta did not. Combinations not including beta were all inactive. 3. The ATPase activity reconstituted from alpha + beta + delta was thermolabile and insensitive to NaN3, whereas the activities obtained from mixtures containing beta and gamma were thermostable and sensitive to NaN3, like the native ATPase. 4. The assemblies containing both beta and gamma subunits had the same mobility as the native ATPase molecule on gel electrophoresis, those without the gamma subunit moved more rapidly toward the anode. 5. Subunits epsilon and delta did not inhibit the ATPase activity of either the assembly (alpha + beta + gamma) or the native ATPase.     

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.     

Acetylcholine receptor assembly is stimulated by phosphorylation of its gamma subunit

Different combinations of Torpedo acetylcholine receptor (AChR) subunits stably expressed in mouse fibroblasts were used to establish a role for phosphorylation in AChR biogenesis. When cell lines expressing fully functional AChR complexes (alpha 2 beta gamma delta) were labeled with 32P, only gamma and delta subunits were phosphorylated. Forskolin, which causes a 2- to 3-fold increase in AChR expression by stimulating subunit assembly, increased unassembled gamma phosphorylation, but had little effect on unassembled delta. The forskolin effect on subunit phosphorylation was rapid, significantly preceding its effect on expression. The pivotal role of the gamma subunit was established by treating alpha beta gamma and alpha beta delta cell lines with forskolin and observing increased expression of only alpha beta gamma complexes. This effect was also observed in alpha gamma, but not alpha delta cells. We conclude that the cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of unassembled gamma subunits, which leads to increased efficiency of assembly of all four subunits.     

Blue native PAGE as a useful method for the analysis of the assembly of distinct combinations of nicotinic acetylcholine receptor subunits

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma delta > alpha 1 beta 1 gamma > alpha 1 beta 1 delta > alpha 1 gamma delta > alpha 1 delta > alpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.     

Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.     

The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: beta 4

A new nicotinic acetylcholine receptor (nAChR) subunit, beta 4, was identified by screening a rat genomic library. In situ hybridization histochemistry revealed expression of the beta 4 gene in the medial habenula of adult rat brains. The primary structure of this subunit was deduced from a cDNA clone isolated from a PC12 cDNA library. Functional nAChRs were detected in Xenopus oocytes injected in pairwise combinations with in vitro synthesized RNAs encoding beta 4 and either the alpha 2, alpha 3, or alpha 4 subunit. Unlike the alpha 3 beta 2 receptor, the alpha 3 beta 4 receptor is not blocked by bungarotoxin 3.1, indicating that the beta subunit can affect the sensitivity of neuronal nAChRs to this toxin. These results extend the functional diversity of nicotinic receptors in the nervous system.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Functional properties of recombinant rat GABAA receptors depend upon subunit composition.

GABA-gated chloride channels were expressed in human embryonic kidney cells following transfection of cDNAs encoding the alpha 1, beta 2, and gamma 2 subunits of the rat GABAA receptor (GABAR). Functional properties were determined using patch-clamp techniques in the whole-cell and outside-out configurations. Large whole-cell currents were observed in cells expressing the alpha 1 beta 2, alpha 1 gamma 2, and alpha 1 beta 2 gamma 2 subunit combinations. The unique characteristics of GABAR channels consisting of these subunit combinations depended upon the presence or absence of beta 2 and gamma 3 subunits. GABA-activated currents in cells expressing GABARs with the beta 2 subunit desensitized faster and showed greater outward rectification, and the channels had a shorter mean open time than GABARs composed of alpha 1 gamma 2 subunits. When the gamma 2 subunit was present the resulting GABAR channels had a larger conductance. The slope of the concentration-response curve was significantly steeper for GABARs composed of alpha 1 beta 2 gamma 2 subunits compared with GABARs consisting of alpha 1 beta 2 or alpha 1 gamma 2 subunit combinations.     

alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.     

Assembly intermediates of the mouse muscle nicotinic acetylcholine receptor in stably transfected fibroblasts

We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor alpha and gamma, and alpha and delta subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing alpha and delta subunits are formed in a time-dependent manner. The alpha and gamma- and the alpha and delta-producing transfected cells form complexes that, when labeled with 125I-alpha- bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the alpha and gamma subunits expressed in fibroblasts suggesting that gamma may have more than one binding site for alpha subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-alpha- bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast delta subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, alpha gamma and alpha delta complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing alpha and gamma or alpha and delta subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.     

Dihydropyridine binding of the calcium channel complex from skeletal muscle is modulated by subunit interaction.

The dihydropyridine-binding subunit alpha 1 of the calcium channel complex from rabbit skeletal muscle can be partially depleted from the alpha 2 delta beta-complex using wheat germ agglutinin-affinity chromatography. This depletion of the alpha 1 from the other subunits leads to a loss of dihydropyridine-binding, which can be fully reconstituted by repletion of the alpha 1 with the other subunits. Reassembly of these subunits results in an increase in the Kd and Bmax of the dihydropyridine-binding indicating that the non-dihydropyridine-binding subunits influence dihydropyridine-binding. The affinity of the alpha 1 subunit for the other subunits was determined to be approximately 35 nM. Since the free alpha 1 subunit will not bind to the beta subunit alone, there is evidence, given the selective partitioning of the beta subunit to the lectin-bound subunit pool, that either beta binds with higher affinity to the alpha 2 delta-complex than to the free alpha 1 subunit or that the bound alpha 1 creates or modulates beta-binding. This indicates a functional high affinity interaction between the dihydropyridine-binding alpha 1 subunit and the alpha 2 delta beta-complex.     

Auxiliary subunits: essential components of the voltage-gated calcium channel complex

Voltage-gated calcium channels are important mediators of several physiological processes, including neuronal excitability and muscle contraction. At the molecular level, the channels are composed of four subunits--the pore forming alpha(1) subunit and the auxiliary alpha(2)delta, beta and gamma subunits. The auxiliary subunits modulate the trafficking and the biophysical properties of the alpha(1) subunit. In the past several years there has been an acceleration of our understanding of the auxiliary subunits, primarily because of their molecular characterization and the availability of spontaneous and targeted mouse mutants. These studies have revealed the crucial role of the subunits in the functional effects that are mediated by voltage-gated calcium channels.