Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma

The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.     

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.     

Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.     

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.     

Cloning and functional characterisation of two novel nicotinic acetylcholine receptor alpha subunits from the insect pest Myzus persicae

Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor alpha subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mp alpha1 and Mp alpha2, are respective structural homologues of the Drosophila D alpha2/Schistocerca gregaria alphaL1 alpha-subunit pair and the Drosophila ALS alpha subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick beta2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mp alpha2 and none with Mp alpha1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor alpha subunits from different species can exhibit distinctive physiological and pharmacological characteristics.     

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.     

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.     

Stable restoration of pyruvate dehydrogenase complex in E1-defective human lymphoblastoid cells: evidence that three C-terminal amino acids of E1 alpha are essential for the structural integrity of heterotetrameric E1

In an attempt to restore pyruvate dehydrogenase complex (PDHC), expression vectors carrying wildtype E1 alpha cDNA (pRAWT) or 1162ins-mutant (pRA1162) were introduced into human lymphoblastoid cells which had a 4-bp insertion after nucleotide 1162 (1162ins) of E1 alpha cDNA, 28% of normal PDHC activity, and undetectable levels of both E1 alpha and E1 beta proteins. The amount of E1 alpha mRNA transcribed from the introduced cDNA was approximately 25 times greater than that transcribed from the endogenous gene. The PDHC activity of pRAWT-transformed cells increased to the normal level whereas this activity increased to 55% of the control in pRA1162-transformed cells. Mitochondria from pRAWT-transformed cells contained normal amounts of both the E1 alpha and the E1 beta subunits. These results suggest that the three C-terminal amino acids of E1 alpha, which were absent from 1162ins-mutant protein, may be important for the structural integrity of E1 and that a large amount of normal subunit, compared to the endogenous mutant enzyme, must be expressed to restore a multienzyme complex.     

The alpha1 and alpha2 isoforms of the AMP-activated protein kinase have similar activities in rat liver but exhibit differences in substrate specificity in vitro

The AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of a catalytic subunit (alpha) and two regulatory subunits (beta and gamma). Two isoforms of the catalytic subunit (alpha1 and alpha2) have been identified. We show here that the alpha1- and alpha2-containing complexes contribute approximately equally to total AMPK activity in rat liver. Furthermore, expression of alpha1 or alpha2 with beta and gamma in mammalian cells demonstrates that both complexes have equal specific activity measured with the SAMS peptide. Using variant peptides, however, we show that alpha1 and alpha2 exhibit slightly different substrate preferences, which suggest that the two isoforms could play different physiological roles within the cell.     

Modification of domains of alpha and beta subunits of F1-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms, by 3''-O-(4-benzoyl)benzoyl adenosine 5''-triphosphate (BzATP)

Photoaffinity labeling by 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed alpha and beta subunits of F1-ATPase from the thermophilic bacterium PS3 (TF1) is described. BzATP binds to both isolated alpha and beta subunits, to complexed beta subunit but not to complexed alpha subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [gamma-32P]BzATP-labeled alpha subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422-Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably alpha Q422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed beta subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments beta G10-M64, beta E75-M233, and beta D390-M469 were labeled with the isolated beta subunit. With complexed beta subunit the label was found only in CNBr fragments: beta E75-M233 and beta G339-M389. The locations where the covalently bound BzATP was found, in the soluble and assembled subunits, indicate that different conformational states exist. In the isolated form of the alpha and beta subunits the amino- and carboxy-termini can fold and reach the central domain of the polypeptide, the domain containing the adenine nucleotide binding site. When alpha combines with beta to form the alpha 3 beta 3 core complex the new conformation of the subunits is such that covalent labeling by BzATP of alpha and of the amino terminal of beta subunit is excluded.     

Wild isolates of murine cytomegalovirus induce myocarditis and antibodies that cross-react with virus and cardiac myosin

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic metabolism of the parasitic nematode Ascaris suum. Two isoforms of the alpha-subunit of pyruvate dehydrogenase (E1) have been identified: alpha I is most abundant in anaerobic adult muscle and alpha II in aerobic larvae. Both isoforms have been expressed as alpha 2 beta 2 tetramers with a muscle-specific beta-subunit, purified to apparent homogeneity, reconstituted with E1-deficient adult A. suum muscle PDC, and assayed for PDC and E1 kinase activity. Recombinant alpha II is a poor substrate for the adult E1 kinase, but its stoichiometry of phosphorylation/inactivation is similar to that reported for the human E1. Initially, inactivation parallels the incorporation of about 1 mol 32P/mol E1 and at maximal phosphorylation about 2.4 32P/mol E1 is incorporated. In contrast, recombinant alpha I (r alpha I) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivation. To examine this altered pattern of phosphorylation, the two phosphorylation sites in each E1 alpha subunit of the r alpha I (site 1 and site 2) were changed either individually or together from Ser to Ala by site-directed mutagenesis. Site 1 was phosphorylated more rapidly than site 2, but the phosphorylation of either site resulted in inactivation, and the phosphorylation of only a single E1 alpha subunit of the tetramer was necessary for inactivation. However, both E1 alpha subunits of the tetramer were phosphorylated, based on the incorporation of about 3.5 mol 32P/mol E1 at maximal phosphorylation and the altered mobility of most of the E1 alpha subunits during SDS-PAGE. These observations suggest that the regulation of both E1 isoforms is modified to maintain PDC activity during the transition to anaerobiosis.     

Determinant for beta-subunit regulation in high-conductance voltage-activated and Ca(2+)-sensitive K+ channels: an additional transmembrane region at the N terminus

The pore-forming alpha subunit of large conductance voltage- and Ca(2+)-sensitive K (MaxiK) channels is regulated by a beta subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming alpha subunit necessary for beta-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to beta-subunit modulation, and analyzed the topology of the alpha subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel alpha subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers beta-subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.     

The human glycine receptor beta subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.