Auxiliary Beta Subunits Research Articles

Critical regulation of the voltage-gated sodium channel, Nav1.4, by PIP2.

Voltage-gated sodium (NaV) channels are vital to the electrical signalling of cells. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting a timer on the excitability of tissues. NaV channels are protein complexes that consist of a large, pore forming alpha subunit and are often associated with auxiliary beta subunits. The alpha subunit is made up of four tandem repeat domains (DI-IV) that consist of six transmembrane segments (S1-S6) with S4 in each domain serving as its putative voltage sensor. The gating cycle of NaV channels is subject to tight regulation, with dysfunction leading to several disease states. Most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate [PI(4,5)P2]; however, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Of the nine (NaV1.1 - NaV1.9) family members that have been identified and classified based on tissue distribution, we studied NaV1.4 which is the isoform primarily expressed in skeletal muscle. We used the optogenetically activated inositide phosphatase, pseudojanin, an enzymatic chimera of inositol polyphosphate 5 phosphatase E (INPP5E), which converts PI(4,5)P2 to PI4P and the S. cerevisiae sac1 phosphatase, which dephosphorylates PI(4)P, to evaluate the role of PI(4,5)P2 in NaV1.4 channel gating and regulation. Through a combination of patch-clamp recording and optogenetics we show that dephosphorylating PI(4,5)P2 shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials. PI(4,5)P2 depletion also augments the late current that persists after fast inactivation, a phenotype that is associated with several mutations that lead to myotonia. Through this work, we provide evidence that PI(4,5)P2 is a necessary co-factor in the gating of NaV1.4 channels.

Auxiliary Beta Subunits are not Obligatory for CaV1.3 Function

Auxiliary Beta Subunits are not Obligatory for CaV1.3 Function

Modulation of BK Channel Function by Auxiliary Beta and Gamma Subunits.

The large-conductance, Ca(2+)- and voltage-activated K(+) (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1-β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings.

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart.

Duchenne muscular dystrophy is a fatal X-linked disease characterized by the absence of dystrophin. Approximately 20% of boys will die of dilated cardiomyopathy that is associated with cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. However, the mechanisms for altered energy metabolism are not yet fully clarified. Calcium influx through the L-type Ca(2+) channel is critical for maintaining cardiac excitation and contraction. The L-type Ca(2+) channel also regulates mitochondrial function and metabolic activity via transmission of movement of the auxiliary beta subunit through intermediate filament proteins. Here, we find that activation of the L-type Ca(2+) channel is unable to induce increases in mitochondrial membrane potential and metabolic activity in intact cardiac myocytes from the murine model of Duchenne muscular dystrophy (mdx) despite robust increases recorded in wt myocytes. Treatment of mdx mice with morpholino oligomers to induce exon skipping of dystrophin exon 23 (that results in functional dystrophin accumulation) or application of a peptide that resulted in block of voltage-dependent anion channel (VDAC) "rescued" mitochondrial membrane potential and metabolic activity in mdx myocytes. The mitochondrial VDAC coimmunoprecipitated with the L-type Ca(2+) channel. We conclude that the absence of dystrophin in the mdx ventricular myocyte leads to impaired functional communication between the L-type Ca(2+) channel and mitochondrial VDAC. This appears to contribute to metabolic inhibition. These findings provide new mechanistic and functional insight into cardiomyopathy associated with Duchenne muscular dystrophy.

Identification of beta-2 as a key cell adhesion molecule in PCa cell neurotropic behavior: a novel ex vivo and biophysical approach.

Prostate cancer (PCa) is believed to metastasize through the blood/lymphatics systems; however, PCa may utilize the extensive innervation of the prostate for glandular egress. The interaction of PCa and its nerve fibers is observed in 80% of PCa and is termed perineural invasion (PNI). PCa cells have been observed traveling through the endoneurium of nerves, although the underlying mechanisms have not been elucidated. Voltage sensitive sodium channels (VSSC) are multimeric transmembrane protein complexes comprised of a pore-forming α subunit and one or two auxiliary beta (β) subunits with inherent cell adhesion molecule (CAM) functions. The beta-2 isoform (gene SCN2B) interacts with several neural CAMs, while interacting putatively with other prominent neural CAMs. Furthermore, beta-2 exhibits elevated mRNA and protein levels in highly metastatic and castrate-resistant PCa. When overexpressed in weakly aggressive LNCaP cells (2BECFP), beta-2 alters LNCaP cell morphology and enhances LNCaP cell metastasis associated behavior in vitro. We hypothesize that PCa cells use beta-2 as a CAM during PNI and subsequent PCa metastasis. The objective of this study was to determine the effect of beta-2 expression on PCa cell neurotropic metastasis associated behavior. We overexpressed beta-2 as a fusion protein with enhanced cyan fluorescence protein (ECFP) in weakly aggressive LNCaP cells and observed neurotropic effects utilizing our novel ex vivo organotypic spinal cord co-culture model, and performed functional assays with neural matrices and atomic force microscopy. With increased beta-2 expression, PCa cells display a trend of enhanced association with nerve axons. On laminin, a neural CAM, overexpression of beta-2 enhances PCa cell migration, invasion, and growth. 2BECFP cells exhibit marked binding affinity to laminin relative to LNECFP controls, and recombinant beta-2 ectodomain elicits more binding events to laminin than BSA control. Functional overexpression of VSSC beta subunits in PCa may mediate PCa metastatic behavior through association with neural matrices.

Childhood Sweetheart vs Late Suitor: CaV Channel Regulation by Auxiliary Beta and Alpha2Delta Subunits

High-voltage-activated calcium (CaV) channels link electrical signals to the inflow of Ca2+ ions that trigger biological responses in excitable cells. CaV channels are macromolecular complexes containing pore-forming alpha1 proteins minimally assembled with auxiliary beta and alpha2delta subunits. Both CaVbeta and alpha2delta subunits augment CaV channel currents by enhancing alpha1 trafficking to the cell surface. Whereas CaVbeta (the childhood sweetheart) associates early with alpha1 in the endoplasmic reticulum and promotes forward trafficking, alpha2delta (the late suitor) enhances CaV surface density by stabilizing mature channels at the plasma membrane. Beyond their direct regulatory effects on CaV channels, CaVbeta and alpha2delta are the physical targets for powerful channel modulation by small monomeric G-proteins (RGK) and the anti-epileptic drug, gabapentin, respectively. I will discuss our findings on the different determinants and mechanisms underlying CaV channel regulation by CaVbeta and alpha2delta subunits, and implications of the work for physiological and therapeutic modulation of CaV channels.

Examining Efficacy of “TAT-less” Delivery of a Peptide against the L-Type Calcium Channel in Cardiac Ischemia–Reperfusion Injury

Increased calcium influx through the L-type Ca(2+) channel or overexpression of the alpha subunit of the channel induces cardiac hypertrophy. Cardiac hypertrophy results from increased oxidative stress and alterations in cell calcium levels following ischemia-reperfusion injury and is an independent risk factor for increased morbidity and mortality. We find that decreasing the movement of the auxiliary beta subunit with a peptide derived against the alpha-interacting domain (AID) of the channel attenuates ischemia-reperfusion injury. We compared the efficacy of delivering the AID peptide using a trans-activator of transcription (TAT) sequence with that of the peptide complexed to multifunctional polymeric nanoparticles. The AID-tethered nanoparticles perfused through the myocardium more diffusely and associated with cardiac myocytes more rapidly than the TAT-labeled peptide but had similar effects on intracellular calcium levels. The AID-complexed nanoparticles resulted in a similar reduction in release of creatine kinase and lactate dehydrogenase after ischemia-reperfusion to the TAT-labeled peptide. Since nanoparticle delivery also holds the potential for dual drug delivery, we conclude that AID-complexed nanoparticles may provide an effective platform for peptide delivery in cardiac ischemia-reperfusion injuries.

"Must Channels Open Before Inactivating?" Redux

Voltage-gated ion channels may adopt four distinct conformations - resting, activated, open and inactivated - and can achieve inactivation from either the closed (activated) or open states (CSI or OSI, respectively). Kv4.x channels are subthreshold-operating K+ channels exhibiting A-type, rapidly-inactivating phenotype, and are responsible for the Ito in the heart and ISA in the nervous system. While previous studies from our lab and others strongly suggest CSI in Kv4.x channels, there is no conclusive evidence for this autoregulatory pathway; and the molecular mechanism of CSI remains unsolved. To shed light on the inactivation pathways of native Kv4.x channels, we expressed the ternary Kv4.2 channel complex including auxiliary Beta-subunits (KChIP and DPP6) in mammalian tsA-201 cells and investigated the resulting A-type currents in the whole-cell configuration of the patch-clamp method. Specifically, we implemented model-independent kinetic analyses to test whether the channels must open before they inactivate (Bean. Biophys J 35, 595-614, 1981; Horn, et al. Nature 291, 426-427, 1981). The main results demonstrate that the development of inactivation induced by a conditioning voltage pulse begins without a significant lag and follows an exponential profile. Consequently, this time course does not reflect the trajectory of the change in open probability induced by a step depolarization to the same voltage. Furthermore, contrary to a prediction of conventional OSI, the rate of macroscopic inactivation slows gradually as the membrane is progressively depolarized. The latter indicates that as the voltage-dependent open probability increases, the rate of inactivation decreases. These observations yield strong model-independent support for a mechanism of preferential CSI in the Kv4.2 channel complex. We are testing this hypothesis at the unitary current level and probing the pathways of inactivation in neuronal Kv4.x channels (cerebellar granule and primary sensory neurons). Supported by NIH grant R01 NS032337 (MC).

Examining the Efficacy of a Peptide Derived Against the Alpha-Interacting Domain of the L-type Calcium Channel in Preventing Ischaemia–Reperfusion Injury

The L-type Ca2+ channel is the main route for calcium influx into cardiac myocytes. Increases in intracellular calcium and oxidative stress are involved in the pathophysiology of cardiac hypertrophy. We and others have demonstrated that increased influx through the L-type Ca2+ channel or over-expression of the alpha subunit of the channel induces cardiac hypertrophy. Genetics suppression of the auxiliary beta subunit is effective at regressing cardiac hypertrophy. We hypothesised that decreasing the movement of the auxiliary beta subunit of the L-type Ca2+ channel with a peptide derived against the alpha-interacting domain (AID) of the channel, would attenuate the myocardial damage associated with reperfusion after ischaemia. We isolated hearts from guinea-pigs and perfused the hearts retrogradely on a Langendorff apparatus. Hearts were perfused with Ca2+-containing Krebs solution for 30 minutes, followed by no-flow ischemia for 30 minutes, then reperfusion for 30 minutes in the absence or presence of 1 μM AID-TAT peptide. Damage was assessed as release of creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate. Oxidative stress was assessed as the ratio of reduced to oxidised glutathione (GSH/GSSG). Creatine kinase release was 35.4% less (22.1 ± 13.4 vs. 34.2 ± 9.7 units/L; n = 4) and LDH release was 89.3% less (2.8 ± 0.8 vs. 26.1 ± 11.6 units/L normalised to pre ischaemia-reperfusion; n = 4) in hearts exposed to AID-TAT peptide while GSH/GSSG ratio was largely unchanged. These data suggest that modulating the movement of the auxiliary beta subunit of the L-type Ca2+ channel may represent a means for reducing myocardial damage during reperfusion.

Evolutionary coupling in the KV1.2-β2 complex

KV1.2 is a potassium channel protein whose electrophysiological properties, including an oxygen sensing response, are modulated by auxiliary beta subunits. The beta subunits are homologous to oxidoreductases, supporting a hypothesis that the coupling of the two subunits helps connect the redox state of the cell to the electrical activity of the membrane. However the exact mechanism of the coupling has not been discovered to-date. We apply evolutionary correlation analysis to infer previously unknown components of the interaction network regulating the response to hypoxia of the KV1.2/β2 complex. Briefly, evolutionary correlation analysis involves finding correlated amino acid substitutions in functionally equivalent proteins (for both subunits) across a range of species. The method thus depends on a reliable method of inferring functionally equivalent (orthologous) proteins in different species, which method we describe in a paper recently published in PLoS ONE. One key finding is the characterization of a network of motif interactions between the α and the β subunits. By significance testing, we show that the likelihood of the correlations shown in the KV1.2-β2 two interaction motifs arising by chance is less than 0.0003, which shows that the correlations are statistically highly significant. We therefore believe that the correlations are likely to be biologically relevant. Other major findings are correlations between specific motifs in the KV1.2-β2 complex and motifs in other proteins such as RACK1 and Eif36sip, which in turn are connected to the hypoxia response. Our paper combines this correlation with the literature evidence on potassium channel inactivation and hypoxia response to identify specific motifs to serve as experimental targets for studies focused on this response. This work aims to add to our general understanding of two major issues in ion channel science: 1) how multi-protein complexes including ion channels function in coordinated fashion, and 2) how ion channels mediate the conversation between the intracellular and extracellular environments. We also aim to apply to ion channel science the principle that domain-domain interactions in proteins can be inferred from correlation of amino-acid substitutions in sets of functionally equivalent proteins.

LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium

Large-conductance, voltage- and calcium-activated potassium (BK, or K(Ca)1.1) channels are ubiquitously expressed in electrically excitable and non-excitable cells, either as alpha-subunit (BKalpha) tetramers or together with tissue specific auxiliary beta-subunits (beta1-beta4). Activation of BK channels typically requires coincident membrane depolarization and elevation in free cytosolic Ca(2+) concentration ([Ca(2+)](i)), which are not physiological conditions for most non-excitable cells. Here we present evidence that in non-excitable LNCaP prostate cancer cells, BK channels can be activated at negative voltages without rises in [Ca(2+)](i) through their complex with an auxiliary protein, leucine-rich repeat (LRR)-containing protein 26 (LRRC26). LRRC26 modulates the gating of a BK channel by enhancing the allosteric coupling between voltage-sensor activation and the channel's closed-open transition. This finding reveals a novel auxiliary protein of a voltage-gated ion channel that gives an unprecedentedly large negative shift ( approximately -140 mV) in voltage dependence and provides a molecular basis for activation of BK channels at physiological voltages and calcium levels in non-excitable cells.

BKbeta1 subunits contribute to BK channel diversity in rat hypothalamic neurons.

Large conductance Ca(2+)-activated BK channels are important regulators of action potential duration and firing frequency in many neurons. As the pore-forming subunits of BK channels are encoded by a single gene, channel diversity is mainly generated by alternative splicing and interaction with auxiliary beta-subunits (BKbeta1-4). In hypothalamic neurons several BK channel subtypes have been described electrophysiologically; however, the distribution of BKbeta subunits is unknown so far. Therefore, an antibody against the large extracellular loop of the BKbeta1 subunit was raised, freed from cross-reactivity against BKbeta2-4 and affinity-purified. The resulting polyclonal monospecific BKbeta1 antibody was characterized by Western blot analysis, ELISA techniques and immunocytochemical staining of BKbeta1-4-transfected CHO and COS-1 cells. Regional and cellular distribution in the rat hypothalamus was analysed by immunocytochemistry and in situ hybridization experiments. Immunocytochemical staining of rat hypothalamic neurons indicates strong BKbeta1 expression in the supraoptic nucleus and the magno- and parvocellular parts of the paraventricular nucleus. Lower expression was found in periventricular nucleus, the arcuate nucleus and in the median eminence. Immunostaining was predominantly localized to somata. In addition, pericytes and ependymal epithelial cells showed BKbeta1 labelling. In all cases immunocytochemical results were supported by in situ hybridization.

Loss-of-function mutation of the SCN3B-encoded sodium channel 3 subunit associated with a case of idiopathic ventricular fibrillation

Loss-of-function mutations in the SCN5A-encoded sodium channel SCN5A or Nav1.5 have been identified in idiopathic ventricular fibrillation (IVF) in the absence of Brugada syndrome phenotype. Nav1.5 is regulated by four sodium channel auxiliary beta subunits. Here, we report a case with IVF and a novel mutation in the SCN3B-encoded sodium channel beta subunit Navbeta3 that causes a loss of function of Nav1.5 channels in vitro. Comprehensive open reading frame mutational analysis of KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, GPD1L, four sodium channel beta subunit genes (SCN1-4B), and targeted scan of RYR2 was performed. A novel missense mutation, Navbeta3-V54G, was identified in a 20-year-old male following witnessed collapse and defibrillation from VF. The ECG exhibited epsilon waves, and imaging studies demonstrated a structurally normal heart. The mutated residue was highly conserved across species, localized to the Navbeta3 extracellular domain, and absent in 800 reference alleles. We found that HEK-293 cells had endogenous Navbeta3, but COS cells did not. Co-expression of Nav1.5 with Navbeta3-V54G (with or without co-expression of the Navbeta1 subunit) in both HEK-293 cells and COS cells revealed a significant decrease in peak sodium current and a positive shift of inactivation compared with WT. Co-immunoprecipitation experiments showed association of Navbeta3 with Nav1.5, and immunocytochemistry demonstrated a dramatic decrease in trafficking to the plasma membrane when co-expressed with mutant Navbeta3-V54G. This study provides molecular and cellular evidence implicating mutations in Navbeta3 as a cause of IVF.

KCNE2 modulation of Kv4.3 current and its potential role in fatal rhythm disorders

The transient outward current I(to) is of critical importance in regulating myocardial electrical properties during the very early phase of the action potential. The auxiliary beta subunit KCNE2 recently was shown to modulate I(to). The purpose of this study was to examine the contributions of KCNE2 and its two published variants (M54T, I57T) to I(to). The functional interaction between Kv4.3 (alpha subunit of human I(to)) and wild-type (WT), M54T, and I57T KCNE2, expressed in a heterologous cell line, was studied using patch-clamp techniques. Compared to expression of Kv4.3 alone, co-expression of WT KCNE2 significantly reduced peak current density, slowed the rate of inactivation, and caused a positive shift of voltage dependence of steady-state inactivation curve. These modifications rendered Kv4.3 channels more similar to native cardiac I(to). Both M54T and I57T variants significantly increased I(to) current density and slowed the inactivation rate compared with WT KCNE2. Moreover, both variants accelerated the recovery from inactivation. The study results suggest that KCNE2 plays a critical role in the normal function of the native I(to) channel complex in human heart and that M54T and I57T variants lead to a gain of function of I(to), which may contribute to generating potential arrhythmogeneity and pathogenesis for inherited fatal rhythm disorders.

Activity and calcium regulate nuclear targeting of the calcium channel beta4b subunit in nerve and muscle cells

Auxiliary beta subunits are critical determinants of membrane expression and gating properties of voltage-gated calcium channels. Mutations in the beta4 subunit gene cause ataxia and epilepsy. However, the specific function of beta4 in neurons and its causal relation to neurological diseases are unknown. Here we report the localization of the beta4 subunit in the nuclei of cerebellar granule and Purkinje cells. beta4b was the only beta isoform showing nuclear targeting when expressed in neurons and skeletal myotubes. Its specific nuclear targeting property was mapped to an N-terminal double-arginine motif, which was necessary and sufficient for targeting beta subunits into the nucleus. Spontaneous electrical activity and calcium influx negatively regulated beta4b nuclear localization by a CRM-1-dependent nuclear export mechanism. The activity-dependent shuttling of beta4b into and out of the nucleus indicates a specific role of this beta subunit in neurons, in communicating the activity of calcium channels to the nucleus.

Intracellular domains interactions and gated motions of IKS potassium channel subunits

Voltage-gated K(+) channels co-assemble with auxiliary beta subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 beta subunits generates the repolarizing K(+) current I(KS). However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life-threatening long or short QT syndromes. Here, we studied the interactions and voltage-dependent motions of I(KS) channel intracellular domains, using fluorescence resonance energy transfer combined with voltage-clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C-terminus interacts with the coiled-coil helix C of the Kv7.1 tetramerization domain. This association is important for I(KS) channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant-negative C-terminal domain. On channel opening, the C-termini of Kv7.1 and KCNE1 come close together. Co-expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K(+) currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C-termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.

Voltage-gated Sodium Channel Activity Promotes Cysteine Cathepsin-dependent Invasiveness and Colony Growth of Human Cancer Cells

Voltage-gated sodium channels (Na(V)) are functionally expressed in highly metastatic cancer cells derived from nonexcitable epithelial tissues (breast, prostate, lung, and cervix). MDA-MB-231 breast cancer cells express functional sodium channel complexes, consisting of Na(V)1.5 and associated auxiliary beta-subunits, that are responsible for a sustained inward sodium current at the membrane potential. Although these channels do not regulate cellular multiplication or migration, their inhibition by the specific blocker tetrodotoxin impairs both the extracellular gelatinolytic activity (monitored with DQ-gelatin) and cell invasiveness leading to the attenuation of colony growth and cell spreading in three-dimensional Matrigel-composed matrices. MDA-MB-231 cells express functional cysteine cathepsins, which we found play a predominant role ( approximately 65%) in cancer invasiveness. Matrigel invasion is significantly decreased in the presence of specific inhibitors of cathepsins B and S (CA-074 and Z-FL-COCHO, respectively), and co-application of tetrodotoxin does not further reduce cell invasion. This suggests that cathepsins B and S are involved in invasiveness and that their proteolytic activity partly depends on Na(V) function. Inhibiting Na(V) has no consequence for cathepsins at the transcription, translation, and secretion levels. However, Na(V) activity leads to an intracellular alkalinization and a perimembrane acidification favorable for the extracellular activity of these acidic proteases. We propose that Na(v) enhance the invasiveness of cancer cells by favoring the pH-dependent activity of cysteine cathepsins. This general mechanism could lead to the identification of new targets allowing the therapeutic prevention of metastases.

Late sodium current is a new therapeutic target to improve contractility and rhythm in failing heart.

Most cardiac Na+ channels open transiently within milliseconds upon membrane depolarization and are responsible for the excitation propagation. However, some channels remain active during hundreds of milliseconds, carrying the so-called persistent or late Na+ current (I(NaL)) throughout the action potential plateau. I(NaL) is produced by special gating modes of the cardiac-specific Na+ channel isoform. Experimental data accumulated over the past decade show the emerging importance of this late current component for the function of both normal and especially failing myocardium, where I(NaL) is reportedly increased. Na+ channels represent a multi-protein complex and its activity is determined not only by the pore-forming alpha subunit but also by its auxiliary beta subunits, cytoskeleton, and by Ca2+ signaling and trafficking proteins. Remodeling of this protein complex and intracellular signaling pathways may lead to alterations of I(NaL) in pathological conditions. Increased I(NaL) and the corresponding Na+ influx in failing myocardium contribute to abnormal repolarization and an increased cell Ca2+ load. Interventions designed to correct I(NaL) rescue normal repolarization and improve Ca2+ handling and contractility of the failing cardiomyocytes. New therapeutic strategies to target both arrhythmias and deficient contractility in HF may not be limited to the selective inhibition of I(NaL) but also include multiple indirect, modulatory (e.g. Ca(2+)- or cytoskeleton- dependent) mechanisms of I(NaL) function.

Structural Basis for Toxin Resistance of β4-Associated Calcium-activated Potassium (BK) Channels

The functional diversity of large conductance Ca(2+)- and voltage-dependent K(+) (BK) channels arises mainly from co-assembly of the pore-forming mSlo alpha subunits with four tissue-enriched auxiliary beta subunits. The structural basis of the interaction between alpha subunits with beta subunits is not well understood. Using computational and experimental methods, we demonstrated that four mSlo turrets decentralized distally from the channel pore to provide a wide open conformation and that the mSlo and hbeta4 subunits together formed a "helmet" containing three basic residues (Lys-120, Arg-121, and Lys-125), which impeded the entry of charybdotoxin (ChTX) by both the electrostatic interaction and limited space. In addition, the tyrosine insert mutant (in100Y) showed 56% inhibition, with a K(d) = 17 nm, suggesting that the hbeta4 lacks an external ChTX-binding site (Tyr-100). We also found that mSlo had an internal binding site (Tyr-294) in the alpha subunits that could "permanently" block 15% of mSlo+hbeta4 currents in the presence of 100 nm ChTX. These findings provide a better understanding of the diverse interactions between alpha and beta subunits and will improve the design of channel inhibitors.

The calcium channel β2 (CACNB2) subunit repertoire in teleosts

BackgroundCardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary β subunits to chaperone the pore-forming α subunit to the plasma membrane, and to modulate channel electrophysiology [1]. Several β subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse β2, but not in the other three β family members, are embryonic lethal at E10.5 due to defects in cardiac contractility [2]. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of β subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of β2 subunits in zebrafish and other teleosts.ResultsCloning of two zebrafish β2 subunit genes (β2.1 and β2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish β2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but β2.2 is much more divergent in sequence than β2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both β2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single β2 subunit gene loci. Comparative analysis of the teleost and human β2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced β2 transcript variants is detected in the embryonic heart compared to the adult.ConclusionThese studies refine our understanding of β2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of β2 subunit diversity in the embryonic heart.