Calcium channel auxiliary subunits

Many channels and carriers associate with auxiliary subunits which modify their activities and facilitate biogenesis. Advances in genome sequencing as well as biochemical, molecular genetic, and physiological experimentation have allowed for the discovery of many transport auxiliary subunits. Recent interests in the pharmacology of the calcium auxiliary subunits prompted a large amount of effort in deciphering their specific role in the conductance of calcium ions. In this review, we evaluate the functions of the 'extra' subunits of the voltage-gated calcium channels in animals as an example of auxiliary subunits of transporters in general. We discuss the functional data available for each of these subunits, present phylogenetic analyses, and discuss their potential evolutionary origins. Our analyses also reveal novel homologues of these subunits which might be of interest to the community.     

Plasma membrane expression of T-type calcium channel alpha(1) subunits is modulated by high voltage-activated auxiliary subunits

It has been suggested that the auxiliary subunits of high voltage-activated (HVA) calcium channels modulate T-type, low voltage-activated (LVA) calcium channels. Such a regulation has yet to be documented, especially because there has been no biochemical characterization of T-channels. To monitor total protein levels and plasma membrane expression of T-channels in living cells, external epitopes (hemagglutinin, FLAG) were introduced into human recombinant Ca(V)3 channels that were also N-terminally fused to green fluorescent protein. Utilizing Western blot techniques, fluorescence flow cytometry, immunofluorescence, luminometry, and electrophysiology, we describe here that beta(1b) and alpha(2)-delta(1) subunits enhance the level of Ca(V)3 proteins as well as their plasma membrane expression in various expression systems. We also report that, in both Xenopus oocytes and mammalian cells, the alpha(2)-delta(1) subunits increase by at least and beta(1b) 2-fold the current density of Ca(V)3 channels with no change in the electrophysiological properties. Altogether, these data indicate that HVA auxiliary subunits modulate Ca(V)3 channel surface expression, suggesting that the membrane targeting of HVA and LVA alpha(1) subunits is regulated dynamically through the expression of a common set of regulatory subunits.     

Sodium channel beta1 subunits promote neurite outgrowth in cerebellar granule neurons

Many immunoglobulin superfamily members are integral in development through regulation of processes such as growth cone guidance, cell migration, and neurite outgrowth. We demonstrate that homophilic interactions between voltage-gated sodium channel beta1 subunits promote neurite extension in cerebellar granule neurons. Neurons isolated from wild-type or beta1(-/-) mice were plated on top of parental, mock-, or beta1-transfected fibroblasts. Wild-type neurons consistently showed increased neurite length when grown on beta1-transfected monolayers, whereas beta1(-/-) neurons showed no increase compared with control conditions. beta1-mediated neurite extension was mimicked using a soluble beta1 extracellular domain and was blocked by antibodies directed against the beta1 extracellular domain. Immunohistochemical analysis suggests that the beta1 and beta4 subunits, but not beta2 and beta3, are expressed in cerebellar Bergmann glia as well as granule neurons. These results suggest a novel role for beta1 during neuronal development and are the first demonstration of a functional role for sodium channel beta subunit-mediated cell adhesive interactions.     

Ca(2+) channel inactivation heterogeneity reveals physiological unbinding of auxiliary beta subunits.

Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations.     

CIB2 and CIB3 are auxiliary subunits of the mechanotransduction channel of hair cells

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Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel

Highly purified L-type Ca(2+) channel complexes containing all five subunits (alpha(1), alpha(2), beta, gamma, and delta) and complexes of alpha(1)-beta subunits were obtained from skeletal muscle triad membranes by three-step purification and by 1% Triton X-100 treatment, respectively. Their structures and the subunit arrangements were analyzed by electron microscopy. Projection images of negatively stained Ca(2+) channels and alpha(1)-beta complexes were aligned, classified and averaged. The alpha(1)-beta complex showed a hollow trapezoid shape of 12 nm height. In top view, four asymmetric domains surrounded a central depression predicted to form the channel pore. The complete Ca(2+) channel complex exhibited the cylindrical shape of 20 nm in height binding a spherical domain on one edge. Further image analysis of higher complexes of the Ca(2+) channel using a monoclonal antibody against the beta subunit showed that the alpha(1)-beta complex forms the non-decorated side of the cylinder, which can traverse the membrane from outside the cell to the cytoplasm. Based on these results, we propose that the Ca(2+) channel exhibits an asymmetric arrangement of auxiliary subunits.     

Sodium channel beta1 and beta3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain

Sequence homology predicts that the extracellular domain of the sodium channel beta1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that beta1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III-like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of beta1, confirming the proposed function of this domain as a cell adhesion molecule. beta1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and beta1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of beta1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.     

Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact

Sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The auxiliary beta subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. beta1 and beta2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that beta1 and beta2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain beta1 and beta2, but not alphaIIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas alphaIIA subunits expressed alone do not cause cellular aggregation, beta subunits co-expressed with alphaIIA retain the ability to adhere and recruit ankyrin. Truncated beta subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of beta1 and beta2 are required for cytoskeletal interactions. It is hypothesized that sodium channel beta subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier.     

MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: auxiliary subunits that enable channel activity

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.     

A family of gamma-like calcium channel subunits

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.     

Heteromeric assembly of acid-sensitive ion channel and epithelial sodium channel subunits

Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.     

Calcium channel beta subunits differentially modulate recovery of the channel from inactivation

We examined the effects of calcium channel beta subunits upon the recovery from inactivation of alpha(1) subunits expressed in Xenopus oocytes. Recovery of the current carried by the L-type alpha(1) subunit (cyCa(v)1) from the jellyfish Cyanea capillata was accelerated by coexpression of any beta subunit, but the degree of potentiation differed according to which beta isoform was coexpressed. The Cyanea beta subunit was most effective, followed by the mammalian b(3), b(4), and beta(2a) subtypes. Recovery of the human Ca(v)2.3 subunit was also modulated by beta subunits, but was slowed instead. beta(3) was the most potent subunit tested, followed by beta(4), then beta(2a), which had virtually no effect. These results demonstrate that different beta subunit isoforms can affect recovery of the channel to varying degrees, and provide an additional mechanism by which beta subunits can differentially regulate alpha(1) subunits.     

Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels

An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

A novel Ca(V)1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits

Voltage-dependent L-type Ca(2+) (Ca(V)1.2) channels are the principal Ca(2+) entry pathway in arterial myocytes. Ca(V)1.2 channels regulate multiple vascular functions and are implicated in the pathogenesis of human disease, including hypertension. However, the molecular identity of Ca(V)1.2 channels expressed in myocytes of myogenic arteries that regulate vascular pressure and blood flow is unknown. Here, we cloned Ca(V)1.2 subunits from resistance size cerebral arteries and demonstrate that myocytes contain a novel, cysteine rich N terminus that is derived from exon 1 (termed "exon 1c"), which is located within CACNA1C, the Ca(V)1.2 gene. Quantitative PCR revealed that exon 1c was predominant in arterial myocytes, but rare in cardiac myocytes, where exon 1a prevailed. When co-expressed with alpha(2)delta subunits, Ca(V)1.2 channels containing the novel exon 1c-derived N terminus exhibited: 1) smaller whole cell current density, 2) more negative voltages of half activation (V(1/2,act)) and half-inactivation (V(1/2,inact)), and 3) reduced plasma membrane insertion, when compared with channels containing exon 1b. beta(1b) and beta(2a) subunits caused negative shifts in the V(1/2,act) and V(1/2,inact) of exon 1b-containing Ca(V)1.2alpha(1)/alpha(2)delta currents that were larger than those in exon 1c-containing Ca(V)1.2alpha(1)/alpha(2)delta currents. In contrast, beta(3) similarly shifted V(1/2,act) and V(1/2,inact) of currents generated by exon 1b- and exon 1c-containing channels. beta subunits isoform-dependent differences in current inactivation rates were also detected between N-terminal variants. Data indicate that through novel alternative splicing at exon 1, the Ca(V)1.2 N terminus modifies regulation by auxiliary subunits. The novel exon 1c should generate distinct voltage-dependent Ca(2+) entry in arterial myocytes, resulting in tissue-specific Ca(2+) signaling.     

The sigma receptor as a ligand-regulated auxiliary potassium channel subunit

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.     

Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits

A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.     

Species-specific Differences among KCNMB3 BK beta3 auxiliary subunits: some beta3 N-terminal variants may be primate-specific subunits

The KCNMB3 gene encodes one of a family of four auxiliary beta subunits found in the mammalian genome that associate with Slo1 alpha subunits and regulate BK channel function. In humans, the KCNMB3 gene contains four N-terminal alternative exons that produce four functionally distinct beta3 subunits, beta3a-d. Three variants, beta3a-c, exhibit kinetically distinct inactivation behaviors. Since investigation of the physiological roles of BK auxiliary subunits will depend on studies in rodents, here we have determined the identity and functional properties of mouse beta3 variants. Whereas beta1, beta2, and beta4 subunits exhibit 83.2%, 95.3%, and 93.8% identity between mouse and human, the mouse beta3 subunit, excluding N-terminal splice variants, shares only 62.8% amino acid identity with its human counterpart. Based on an examination of the mouse genome and screening of mouse cDNA libraries, here we have identified only two N-terminal candidates, beta3a and beta3b, of the four found in humans. Both human and mouse beta3a subunits produce a characteristic use-dependent inactivation. Surprisingly, whereas the hbeta3b exhibits rapid inactivation, the putative mbeta3b does not inactivate. Furthermore, unlike hbeta3, the mbeta3 subunit, irrespective of the N terminus, mediates a shift in gating to more negative potentials at a given Ca(2+) concentration. The shift in gating gradually is lost following patch excision, suggesting that the gating shift involves some regulatory process dependent on the cytosolic milieu. Examination of additional genomes to assess conservation among splice variants suggests that the putative mbeta3b N terminus may not be a true orthologue of the hbeta3b N terminus and that both beta3c and beta3d appear likely to be primate-specific N-terminal variants. These results have three key implications: first, functional properties of homologous beta3 subunits may differ among mammalian species; second, the specific physiological roles of homologous beta3 subunits may differ among mammalian species; and, third, some beta3 variants may be primate-specific ion channel subunits.