Sodium channel auxiliary subunits

Voltage-gated ion channels are well known for their functional roles in excitable tissues. Excitable tissues rely on voltage-gated ion channels and their auxiliary subunits to achieve concerted electrical activity in living cells. Auxiliary subunits are also known to provide functional diversity towards the transport and biogenesis properties of the principal subunits. Recent interests in pharmacological properties of these auxiliary subunits have prompted significant amounts of efforts in understanding their physiological roles. Some auxiliary subunits can potentially serve as drug targets for novel analgesics. Three families of sodium channel auxiliary subunits are described here: beta1 and beta3, beta2 and beta4, and temperature-induced paralytic E (TipE). While sodium channel beta-subunits are encoded in many animal genomes, TipE has only been found exclusively in insects. In this review, we present phylogenetic analyses, discuss potential evolutionary origins and functional data available for each of these subunits. For each family, we also correlate the functional specificity with the history of evolution for the individual auxiliary subunits.     

Phylogenetic analyses of potassium channel auxiliary subunits

Results of recent genome-sequencing projects together with advances in biochemical, molecular genetic and physiological experimentation have allowed discovery of many transport auxiliary subunits. These subunits facilitate the proper movement of substrates across cell membranes. Mutations of any of these subunits can cause catastrophic effects to the transport mechanism and cause certain genetic diseases. Auxiliary subunits of ion channels are of particular interest because of their potential to diversify the transport properties of the principal subunits. Furthermore, ion channel auxiliary subunits may function in the capacity of enhancing surface expression, allowing gating, and providing chaperone-like activities. As a result of their evolutionary histories, these proteins can be grouped exclusively by phylogenetic techniques. Many of these families are found to be restricted to a single kingdom of life while others seem to be ubiquitous. Here we report the results of systematic analyses of three families of ion channel auxiliary subunits. Some subunits contain one or more transmembrane segments while others exist only in the cytoplasm. We have also observed potential horizontal transfer across kingdoms with these auxiliary subunits. In this report, we present tabulated results of homology searches, partial multiple alignments, secondary structure analyses, and phylogenetic trees for these families.     

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.     

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.     

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.     

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.     

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

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Calcium channel function regulated by the SH3-GK module in beta subunits

beta subunits of voltage-gated calcium channels (VGCCs) regulate channel trafficking and function, thereby shaping the intensity and duration of intracellular changes in calcium. beta subunits share limited sequence homology with the Src homology 3-guanylate kinase (SH3-GK) module of membrane-associated guanylate kinases (MAGUKs). Here, we show biochemical similarities between beta subunits and MAGUKs, revealing important aspects of beta subunit structure and function. Similar to MAGUKs, an SH3-GK interaction within beta subunits can occur both intramolecularly and intermolecularly. Mutations that disrupt the SH3-GK interaction in beta subunits alter channel inactivation and can inhibit binding between the alpha(1) and beta subunits. Coexpression of beta subunits with complementary mutations in their SH3 and GK domains rescues these deficits through intermolecular beta subunit assembly. In MAGUKs, the SH3-GK module controls protein scaffolding. In beta subunits, this module regulates the inactivation of VGCCs and provides an additional mechanism for tuning calcium responsiveness.     

Calcium channel beta subunits differentially regulate the inhibition of N-type channels by individual Gbeta isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.     

禾谷缢管蚜钠通道辅助亚基对钠通道基因表达水平及杀虫剂敏感性的影响

【目的】通过分析禾谷缢管蚜Rhopalosiphum padi钠通道辅助亚基对钠通道功能的影响,探究辅助亚基在钠离子通道的门控性质中的作用。【方法】分别显微注射dsRpNa_vH1和dsRpNa_vH2对钠通道基因RpNa_vH1和RpNa_vH2进行RNAi后,采用实时定量PCR(qRT-PCR)技术测定禾谷缢管蚜成蚜5个钠通道辅助亚基基因(RpTEH1,RpTEH2,RpTEH3,RpTEH4和RpTipE)的表达量;利用qRT-PCR技术和杀虫剂生物测定分别测定RNAi干扰RpTipE对禾谷缢管蚜成蚜钠通道及其辅助亚基基因表达量以及LC₅₀浓度高效氯氟氰菊酯敏感性的影响;利用双电压钳技术检测非洲爪蟾Xenopus laevis卵母细胞单独注射果蝇Drosophila钠通道基因DmNa_v22 cRNA及DmNa_v22 cRNA分别与果蝇钠通道辅助亚基基因DmTipE及禾谷缢管蚜钠通道辅助亚基基因RpTEH2,RpTEH3,RpTEH4和R...     

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.     

Calcium channel γ subunits provide insights into the evolution of this gene family

The γ subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct γ subunits have been described from human and/or mouse. The first identified member of this group of proteins, γ₁, is a component of the L-type calcium channel expressed in skeletal muscle. A second member, γ₂, identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel γ subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian γ subunits suggests the following relationship ((((γ₂, γ₃), (γ₄, γ₈)), (γ₅, γ₇)), (γ₁, γ₆)) that indicates that they evolved from a common ancestral γ subunit via gene duplication. Our analysis reveals that the novel γ subunit γ₆ most closely resembles γ₁ and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other γ subunits. Rat γ subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of γ₁ mRNA and the long isoform of γ₆ mRNA is most robust in skeletal muscle, while γ₆ is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the γ subunits, we propose that γ₁ and γ₆ may share common physiological functions distinct from the other homologous γ subunits.     

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.     

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.     

Structure of intact human MCU supercomplex with the auxiliary MICU subunits

Dear Editor,Mitochondrial Ca2+homeostasis regulates energy production,cell division,and cell death.The basic properties of mitochondrial Ca2+uptake have been firmly established.The Ca2+influx is mediated by MCU,driven by membrane potential and using a uniporter mechanism(Vasington and Murphy,1962).Patch-clamp analysis of MCU currents demonstrated that MCU is a channel with exceptionally high Ca2+selectivity(Kirichok et al.,2004).