Biomedical subjects

L Birnbaumer

Publications and source records attributed to L Birnbaumer.

At least 37 records · Page 2Linked to original sources

PubMed1999

Complete reversal of run-down in rabbit cardiac Ca2+ channels by patch-cramming in Xenopus oocytes; partial reversal by protein kinase A.

The rabbit cardiac Ca2+ channel (alpha1C) expressed in Xenopus oocytes exhibited a complete run-down of ionic currents when cell-attached patches were excised. The alpha1C channel was expressed alone or was coexpressed with the accessory beta2a or beta1b subunit. The catalytic subunit of protein kinase A (PKAc) and MgATP were capable of delaying the run-down of single-channel currents. In 33% of the alpha1C patches, and 26% of the alpha1C+beta2a patches, inclusion of PKAc in the bath solution delayed the run-down for a maximum of 20 min. In experiments where PKAc in the bath was not sufficient to delay the run-down of channel activity, insertion of the patch back into the oocyte (patch-cramming) could restore channel activity. Gating currents were also measured in the alpha1C+beta1b channel and were not subject to any run-down, even after the complete run-down of ionic currents. The results presented here reveal that PKAc is capable of delaying the run-down of currents in a subset of patches. The patch-cramming results suggest that a cytoplasmic factor, in addition to phosphorylation of the channel (by PKAc), may be involved in the maintenance of channel activity.

Adenosine Triphosphate↗

PubMed1999

Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein.

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+ channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1beta cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1beta cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of approximately 92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.

Amino Acid Sequence↗

PubMed1999

Roles of Gi and Gq/11 in mediating desensitization of the luteinizing hormone/choriogonadotropin receptor in porcine ovarian follicular membranes.

Although desensitization of most guanine nucleotide-binding (G) protein receptors is triggered by phosphorylation of the receptor, desensitization of the LH/CG receptor (-R) in porcine follicular ovarian membranes appears to be independent of LH/CG-R phosphorylation. We therefore evaluated whether desensitization of the LH/CG-R reflected a direct inhibition of adenylyl cyclase (AC) activity by either the alpha-subunit of Gi or betagamma-subunits derived from any of the membrane G proteins activated in response to LH/CG-R activation or whether desensitization reflected a competition between Gs and a G protein that activated phospholipase C for binding sites on the LH/CG-R. The results showed that follicular membrane AC activity was not inhibited upon activation of the LH/CG-R despite evidence that the ACs in follicular membranes, when maximally activated by forskolin, could be inhibited when membrane G proteins were activated by guanyl-5'-yl imidodiphosphate, and that pertussis toxin pretreatment of membranes raised forskolin-stimulated AC activity, consistent with a tonic inhibition of follicular membrane AC activity. Similarly, agonist-stimulated desensitization of LH/CG-R-stimulated AC activity was not inhibited by pertussis toxin. Therefore, desensitization is not the result of inhibition of AC mediated by an inhibitory Gi subunit. Follicular membrane AC was also not inhibited by Gbetagamma subunits freed with activation of Gs Gq/11, or G13, based on the inabilities of exogenous Gbetagamma to promote desensitization and of a protein that sequesters Gbetagamma to inhibit desensitization. Desensitization was also not inhibited by a Gq/11 C-terminal peptide or antiserum directed toward the C-terminus of Gq/11, nor was it reversed with the addition of Gbetagamma to membranes exhibiting desensitized LH/CG-R, suggesting that desensitization is independent of coupling of the LH/CG-R to Gq/11. These results indicate that agonist-dependent desensitization of LH/CG-R-stimulated AC activity is mediated by a unique mechanism.

Adenylate Cyclase Toxin↗

PubMed1998

Functional interaction between InsP3 receptors and store-operated Htrp3 channels.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

Animals↗

PubMed1998

Functional coupling between human E-type Ca2+ channels and mu opioid receptors expressed in Xenopus oocytes.

Neuronal alpha1E Ca2+ channels were expressed in Xenopus laevis oocytes alone and in combination with the mu opioid receptor. Macroscopic currents were recorded under voltage clamp conditions. The stimulation of the morphine receptor by the synthetic [D-Ala2,N-Me-Phe4,Gly-ol5] enkephalin (DAMGO) produced a 20% reduction in the alpha1E ionic current. This effect was associated with a large change in the decay phase of the Ba2+ current. The effect of 1 microM DAMGO was fully antagonized by the universal mu opioid receptor antagonist naloxone and by the selective antagonist beta-funaltrexamine. The ionic current inhibition induced by DAMGO was partially recovered by preceding strong depolarizations. The injection of the catalytic subunit of pertussis toxin (A-protomer) abolished the effect of DAMGO, suggesting the involvement of a GTP binding protein in the alpha1E modulation. The coexpression of the regulatory beta2a Ca2a channel subunit, together with the alpha1E subunit and the mu opioid receptor, prevented the reduction of the ionic current following the receptor stimulation with DAMGO, whereas the coexpression with the beta3 subunit reduced by approximately 50% the modulatory effect of DAMGO. The effect produced by the stimulation of the opioid receptor could be mimicked by coexpressing the alpha1E channel with the G-protein betagamma subunits.

Analgesics, Opioid↗

PubMed1998

Unique regulatory properties of the type 2a Ca2+ channel beta subunit caused by palmitoylation.

Beta subunits of voltage-gated Ca2+ channels are encoded in four genes and display additional molecular diversity because of alternative splicing. At the functional level, all forms are very similar except for beta2a, which differs in that it does not support prepulse facilitation of alpha1C Ca2+ channels, inhibits voltage-induced inactivation of neuronal alpha1E Ca2+ channels, and is more effective in blocking inhibition of alpha1E channels by G protein-coupled receptors. We show that the distinguishing properties of beta2a, rather than interaction with a distinct site of alpha1, are because of the recently described palmitoylation of cysteines in positions three and four, which also occurs in the Xenopus oocyte. Essentially, all of the distinguishing features of beta2a were lost in a mutant that could not be palmitoylated [beta2a(Cys3,4Ser)]. Because protein palmitoylation is a dynamic process, these findings point to the possibility that regulation of palmitoylation may contribute to activity-dependent neuronal and synaptic plasticity. Evidence is presented that there may exist as many as three beta2 splice variants differing only in their N-termini.

Amino Acid Sequence↗

PubMed1998

The membrane topology of human transient receptor potential 3 as inferred from glycosylation-scanning mutagenesis and epitope immunocytochemistry.

Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel alpha1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2-H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly.

Animals↗

PubMed1998

Multiple neurological abnormalities in mice deficient in the G protein Go.

The G protein Go is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, alpha2-adrenergic, M2 muscarinic, and somatostatin receptors. In vitro, Go is also activated by growth cone-associated protein of Mr 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. To learn about the roles that Go may play in intact cells and whole body homeostasis, we disrupted the gene encoding the alpha subunits of Go in embryonic stem cells and derived Go-deficient mice. Mice with a disrupted alphao gene (alphao-/- mice) lived but had an average half-life of only about 7 weeks. No Goalpha was detectable in homogenates of alphao-/- mice by ADP-ribosylation with pertussis toxin. At the cellular level, inhibition of cardiac adenylyl cyclase by carbachol (50-55% at saturation) was unaffected, but inhibition of Ca2+ channel currents by opioid receptor agonist in dorsal root ganglion cells was decreased by 30%, and in 25% of the alphao-/- cells examined, the Ca2+ channel was activated at voltages that were 13.3 +/- 1.7 mV lower than in their counterparts. Loss of alphao was not accompanied by appearance of significant amounts of active free betagamma dimers (prepulse test). At the level of the living animal, Go-deficient mice are hyperalgesic (hot-plate test) and display a severe motor control impairment (falling from rotarods and 1-inch wide beams). In spite of this deficiency, alphao-/- mice are hyperactive and exhibit a turning behavior that has them running in circles for hours on end, both in cages and in open-field tests. Except for one, all alphao-/- mice turned only counterclockwise. These findings indicate that Go plays a major role in motor control, in motor behavior, and in pain perception and also predict involvement of Go in Ca2+ channel regulation by an unknown mechanism.

Abnormalities, Multiple↗

PubMed1998

Long lasting facilitation of the rabbit cardiac Ca2+ channel: correlation with the coupling efficiency between charge movement and pore opening.

Facilitation of Ca2+ entry into cells enhances Ca(2+)-activated events such as transmitter release and stimulation of second messenger systems. We have found a long lasting prepulse facilitation (up to 3-fold) of the cardiac Ca2+ channel alpha1Cbeta1b that lasts for tens of seconds without altering current kinetics. The voltage- and time-dependence of the installation of facilitation was characterized as well as the time- and use-dependence of the decay of facilitation. The degree of facilitation was correlated with the coupling efficiency between the charge movement and pore opening channels that were poorly coupled prior to facilitation exhibited the largest facilitation.

Animals↗

PubMed1998

Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels.

1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1998

Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells.

hTrp3 is a human homologue of the Drosophila gene responsible for a transient receptor potential (trp) mutation. When stably expressed in HEK293 cells, hTrp3 formed ion channels that were active under resting conditions but could be further stimulated by carbachol or ATP via endogenous muscarinic or purinergic receptors, respectively. Agonist evoked currents reversed polarity near 0 mV in physiological ionic conditions and were associated with a significant increase in the current variance. These results suggest the involvement of a non-selective cation channel with relatively large unitary amplitude. Consistent with this, resolved unitary events had a conductance of approximately 60 pS in the negative voltage range and an extrapolated reversal potential near 0 mV.

Adenosine Triphosphate↗

PubMed1998

Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 cells. Evidence for a non-capacitative Ca2+ entry.

Ca2+ release from its internal stores as a result of activation of phospholipase C is accompanied by Ca2+ influx from the extracellular space. Ca2+ influx channels may be formed of proteins homologous to Drosophila Trp. At least six non-allelic Trp genes are present in the mouse genome. Full-length human, bovine, mouse, and rat cDNAs for Trp1, 3, 4, 6 have been cloned. Expression of these genes in various mammalian cells has provided evidence that Trp proteins form plasma membrane Ca2+-permeant channels that can be activated by an agonist that activates phospholipase C, by inositol 1,4, 5-trisphosphate, and/or store depletion. We have stably expressed human Trp3 (hTrp3) in human embryonic kidney (HEK)293 cells. Measurement of intracellular Ca2+ concentrations in Fura2-loaded cells showed that cell lines expressing hTrp3 have significantly higher basal and agonist-stimulated influxes of Ca2+, Mn2+, Ba2+, and Sr2+ than control cells. The increase in Ca2+ entry attributable to the expression of hTrp3 obtained upon store depletion by thapsigargin was much lower than that obtained by stimulation with agonists acting via a Gq-coupled receptor. Addition of agonists to thapsigargin-treated Trp3 cells resulted in a further increase in the entry of divalent cations. The increased cation entry in Trp3 cells was blocked by high concentrations of SKF 96365, verapamil, La3+, Ni2+, and Gd3+. The Trp3-mediated Ca2+ influx activated by agonists was inhibited by a phospholipase C inhibitor, U73122. We propose that expression of hTrp3 in these cells forms a non-selective cation channel that opens after the activation of phospholipase C but not after store depletion. In addition, a subpopulation of the expressed hTrp3 may form heteromultimeric channels with endogenous proteins that are sensitive to store depletion.

Animals↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Modulation of human neuronal alpha 1E-type calcium channel by alpha 2 delta-subunit.

Calcium channels are composed of a pore-forming subunit, alpha 1, and at least two auxiliary subunits, beta- and alpha 2 delta-subunits. It is well known that beta-subunits regulate most of the properties of the channel. The function of alpha 2 delta-subunit is less understood. In this study, the effects of the calcium channel alpha 2 delta-subunit on the neuronal alpha 1E voltage-gated calcium channel expressed in Xenopus oocytes was investigated without and with simultaneous coexpression of either the beta 1b- or the beta 2a-subunit. Most aspects of alpha 1E function were affected by alpha 2 delta. Thus alpha 2 delta caused a shift in the current-voltage and conductance-voltage curves toward more positive potentials and accelerated activation, deactivation, and the installation of the inactivation process. In addition, the efficiency with which charge movement is coupled to pore opening assessed by determining ratios of limiting conductance to limiting charge movement was decreased by alpha 2 delta by factors that ranged from 1.6 (P < 0.01) for alpha 1E-channels to 3.0 (P < 0.005) for alpha 1E beta 1b-channels. These results indicate that alpha 2 delta facilitates the expression and the maturation of alpha 1E-channels and converts these channels into molecules responding more rapidly to voltage.

Animals↗

PubMed1997

Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein.

Hormonal stimulation of Gq-protein coupled receptors triggers Ca2+ mobilization from internal stores. This is followed by a Ca2+ entry through the plasma membrane. Drosophila Trp and Trpl proteins have been implicated in Ca2+ entry and three mammalian homologues of Drosophila Trp/Trpl, hTrp1, hTrp3 and bTrp4 (also bCCE) have been cloned and expressed. Using mouse brain RNA as template, we report here the polymerase chain reaction-based cloning and functional expression of a novel Trp, mTrp6. The cDNA encodes a protein of 930 amino acids, the sequence of which is 36.8, 36.3, 43.1, 38.6, and 74. 1% identical to Drosophila Trp and Trpl, bovine Trp4, and human Trp1 and Trp3, respectively. Transient expression of mTrp6 in COS.M6 cells by transfection of the full-length mTrp6 cDNA increases Ca2+ entry induced by stimulation of co-transfected M5 muscarinic acetylcholine receptor with carbachol (CCh), as seen by dual wavelength fura 2 fluorescence ratio measurements. The mTrp6-mediated increase in Ca2+ entry activity was blocked by SKF-96365 and La3+. Ca2+ entry activity induced by thapsigargin was similar in COS cells transfected with or without the mTrp6 cDNA. The thapsigargin-stimulated Ca2+ entry could not be further stimulated by CCh in control cells but was markedly increased in mTrp6-transfected cells. Records of whole cell transmembrane currents developed in response to voltage ramps from -80 to +40 mV in control HEK cells and HEK cells stably expressing mTrp6 revealed the presence of a muscarinic receptor responsive non-selective cation conductance in Trp6 cells that was absent in control cells. Our data support the hypothesis that mTrp6 encodes an ion channel subunit that mediates Ca2+ entry stimulated by a G-protein coupled receptor, but not Ca2+ entry stimulated by intracellular Ca2+ store depletion.

Amino Acid Sequence↗

PubMed1997

Direct interaction of gbetagamma with a C-terminal gbetagamma-binding domain of the Ca2+ channel alpha1 subunit is responsible for channel inhibition by G protein-coupled receptors.

Several classes of voltage-gated Ca2+ channels (VGCCs) are inhibited by G proteins activated by receptors for neurotransmitters and neuromodulatory peptides. Evidence has accumulated to indicate that for non-L-type Ca2+ channels the executing arm of the activated G protein is its betagamma dimer (Gbetagamma). We report below the existence of two Gbetagamma-binding sites on the A-, B-, and E-type alpha1 subunits that form non-L-type Ca2+ channels. One, reported previously, is in loop 1 connecting transmembrane domains I and II. The second is located approximately in the middle of the ca. 600-aa-long C-terminal tails. Both Gbetagamma-binding regions also bind the Ca2+ channel beta subunit (CCbeta), which, when overexpressed, interferes with inhibition by activated G proteins. Replacement in alpha1E of loop 1 with that of the G protein-insensitive and Gbetagamma-binding-negative loop 1 of alpha1C did not abolish inhibition by G proteins, but the exchange of the alpha1E C terminus with that of alpha1C did. This and properties of alpha1E C-terminal truncations indicated that the Gbetagamma-binding site mediating the inhibition of Ca2+ channel activity is the one in the C terminus. Binding of Gbetagamma to this site was inhibited by an alpha1-binding domain of CCbeta, thus providing an explanation for the functional antagonism existing between CCbeta and G protein inhibition. The data do not support proposals that Gbetagamma inhibits alpha1 function by interacting with the site located in the loop I-II linker. These results define the molecular mechanism by which presynaptic G protein-coupled receptors inhibit neurotransmission.

Animals↗

PubMed1997

Feedback inhibition of Ca2+ channels by Ca2+ depends on a short sequence of the C terminus that does not include the Ca2+ -binding function of a motif with similarity to Ca2+ -binding domains.

alpha(1C)- and alpha(1E)-based Ca2+ channels differ in that the former are inhibited by Ca2+ entering through its pore, while the latter are not. It has been proposed on the basis of analysis of alpha(1E)/alpha(1C) chimeras that the molecular determinants responsible for Ca2+ inhibition involve both a conserved Ca2+-binding motif (EF hand) plus additional sequences located C-terminal to the EF hand. Through construction of similar alpha(1E)/alpha(1C) chimeras, we transferred Ca2+ inhibition from alpha(1C) to alpha(1E) by replacing a 134-aa segment of alpha(1E) with the homologous 142-aa segment of alpha(1C). This segment is located immediately after the proposed Ca2+ -binding EF hand motif. Replacement of the alpha(1C) EF hand with the corresponding EF hand of alpha(1E) did not interfere with inhibition of alpha(1C) by Ca2+, and a triple mutant of alpha(1C), alpha(1C)[D1535A,E1537A,D1546A], that disrupts the potential Ca2+-coordinating ability of the EF hand continued to be inhibited by Ca2+. These results indicate that a small portion of the alpha(1C) C terminus is essential for inhibition by Ca2+ and place the Ca2+ -binding site anywhere in alpha(1C), with the exception of its EF hand-like motif.

Amino Acid Sequence↗

PubMed1997

A Xenopus oocyte beta subunit: evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit.

Two closely related beta subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus beta antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca2+ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian alpha1 cRNAs (alpha(1C) and alpha(1E)); (ii) coinjection of a Xenopus beta antisense oligonucleotide and excess rat beta cRNA rescued expression of alpha1 Ca2+ channel currents; and (iii) coinjection of mammalian alpha1 cRNA with cRNA encoding either of the two Xenopus beta subunits facilitated both activation and inactivation of Ca2+ channel currents by voltage, as happens with most mammalian beta subunits. The Xenopus beta subunit cDNAs (beta3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 beta subunit. We propose that "alpha1 alone" channels are in fact tightly associated alpha1beta3xo channels, and that effects of exogenous beta subunits are due to formation of higher-order [alpha1beta]beta(n) complexes with an unknown contribution of beta3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition alpha1beta, are [alpha1beta]beta(n) complexes that associate with alpha2delta and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of alpha(1E) have a beta subunit binding domain. This beta binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel alpha1 subunits.

Amino Acid Sequence↗