Isoform-specific effects of the beta2 subunit on voltage-gated sodium channel gating

Voltage-gated sodium channels (Nav) are complex glycoproteins comprised of an alpha subunit and often one to several beta subunits. We have shown that sialic acid residues linked to Nav alpha and beta1 subunits alter channel gating. To determine whether beta2-linked sialic acids similarly impact Nav gating, we co-expressed beta2 with Nav1.5 or Nav1.2 in Pro5 (complete sialylation) and in Lec2 (essentially no sialylation) cells. Beta2 sialic acids caused a significant hyperpolarizing shift in Nav1.5 voltage-dependent gating, thus describing for the first time an effect of beta2 on Nav1.5 gating. In contrast, beta2 caused a sialic acid-independent depolarizing shift in Nav1.2 gating. A deglycosylated mutant, beta(2-DeltaN), had no effect on Nav1.5 gating, indicating further the impact of beta2 N-linked sialic acids on Nav1.5 gating. Conversely, beta(2-DeltaN) modulated Nav1.2 gating virtually identically to beta2, confirming that beta2 N-linked sugars have no impact on Nav1.2 gating. Thus, beta2 modulates Nav gating through multiple mechanisms possibly determined by the associated alpha subunit. Beta1 and beta2 were expressed together with Nav1.5 or Nav1.2 in Pro5 and Lec2 cells. Together beta1 and beta2 produced a significantly larger sialic acid-dependent hyperpolarizing shift in Nav1.5 gating. Under fully sialylating conditions, the Nav1.2.beta1.beta2 complex behaved like Nav1.2 alone. When sialylation was reduced, only the sialic acid-independent depolarizing effects of beta2 on Nav1.2 gating were apparent. Thus, the varied effects of beta1 and beta2 on Nav1.5 and Nav1.2 gating are apparently synergistic and highlight the complex manner, through subunit- and sugar-dependent mechanisms, by which Nav activity is modulated.     

Gating and ionic currents reveal how the BKCa channel's Ca2+ sensitivity is enhanced by its beta1 subunit

Large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels) are regulated by the tissue-specific expression of auxiliary beta subunits. Beta1 is predominantly expressed in smooth muscle, where it greatly enhances the BK(Ca) channel's Ca(2+) sensitivity, an effect that is required for proper regulation of smooth muscle tone. Here, using gating current recordings, macroscopic ionic current recordings, and unitary ionic current recordings at very low open probabilities, we have investigated the mechanism that underlies this effect. Our results may be summarized as follows. The beta1 subunit has little or no effect on the equilibrium constant of the conformational change by which the BK(Ca) channel opens, and it does not affect the gating charge on the channel's voltage sensors, but it does stabilize voltage sensor activation, both when the channel is open and when it is closed, such that voltage sensor activation occurs at more negative voltages with beta1 present. Furthermore, beta1 stabilizes the active voltage sensor more when the channel is closed than when it is open, and this reduces the factor D by which voltage sensor activation promotes opening by approximately 24% (16.8-->12.8). The effects of beta1 on voltage sensing enhance the BK(Ca) channel's Ca(2+) sensitivity by decreasing at most voltages the work that Ca(2+) binding must do to open the channel. In addition, however, in order to fully account for the increase in efficacy and apparent Ca(2+) affinity brought about by beta1 at negative voltages, our studies suggest that beta1 also decreases the true Ca(2+) affinity of the closed channel, increasing its Ca(2+) dissociation constant from approximately 3.7 microM to between 4.7 and 7.1 microM, depending on how many binding sites are affected.     

Profiling the phospho-status of the BKCa channel alpha subunit in rat brain reveals unexpected patterns and complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BK(Ca)) channels are tetramers of alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BK(Ca) channels affinity-purified from rat brain to analyze in vivo BKalpha phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKalpha differentially modulates the voltage- and Ca(2+)-dependence of channel activation. These results demonstrate that the pore-forming subunit of BK(Ca) channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKalpha structure and function.     

BKCa通道的结构与功能

BKCa通道将细胞膜电特性与细胞信号系统联系在一起,在细胞功能实现中起着重要作用。该通道广泛且又较高密度地表达于许多物种的多种组织,其分子结构复杂,丰富的超家族成员具有各自不同的表达分布。BKCa通道的分子结构由α亚单位和β亚单位构成,其中α亚单位形成通道的孔道区和活性调节区域,β亚单位修饰通道活性的调节特性。BKCa通道开放几率大、电导率高、调控位点多,并且不同的超家族成员表现出不同的功能特征,如细胞膜电位感受性、细胞内游离钙离子敏感性等。文章概述BKCa通道的分子结构和功能特征。     

Genetic analysis of the mammalian K+ channel beta subunit Kvbeta 2 (Kcnab2)

Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.     

Endothelial K(+) channel lacks the Ca(2+) sensitivity-regulating beta subunit.

Hyperpolarizing large-conductance, Ca(2+)-activated K(+) channels (BK) are important modulators of vascular smooth muscle and endothelial cell function. In vascular smooth muscle cells, BK are composed of pore-forming alpha subunits and modulatory beta subunits. However, expression, composition, and function of BK subunits in endothelium have not been studied so far. In patch-clamp experiments we identified BK (283 pS) in intact endothelium of porcine aortic tissue slices. The BK opener DHS-I (0.05-0.3 micromol/l), stimulating BK activity only in the presence of beta subunits, had no effect on BK in endothelium whereas the alpha subunit selective BK opener NS1619 (20 micromol/l) markedly increased channel activity. Correspondingly, mRNA expression of the beta subunit was undetectable in endothelium, whereas alpha subunit expression was demonstrated. To investigate the functional role of beta subunits, we transfected the beta subunit into a human endothelial cell line (EA.hy 926). beta subunit expression resulted in an increased Ca(2+) sensitivity of BK activity: the potential of half-maximal activation (V(1/2)) shifted from 73.4 mV to 49.6 mV at 1 micromol/l [Ca(2+)](i) and an decrease of the EC(50) value for [Ca(2+)](i) by 1 microM at +60 mV was observed. This study demonstrates that BK channels in endothelium are composed of alpha subunits without association to beta subunits. The lack of the beta subunit indicates a substantially different channel regulation in endothelial cells compared to vascular smooth muscle cells.     

L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII

L-type Ca(2+) channels (LTCCs) are major entry points for Ca(2+) in many cells. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is associated with cardiac LTCC complexes and increases channel open probability (P(O)) to dynamically increase Ca(2+) current (I(Ca)) and augment cellular Ca(2+) signaling by a process called facilitation. However, the critical molecular mechanisms for CaMKII localization to LTCCs and I(Ca) facilitation in cardiomyocytes have not been defined. We show CaMKII binds to the LTCC beta(2a) subunit and preferentially phosphorylates Thr498 in beta(2a). Mutation of Thr498 to Ala (T498A) in beta(2a) prevents CaMKII-mediated increases in the P(O) of recombinant LTCCs. Moreover, expression of beta(2a)(T498A) in adult cardiomyocytes ablates CaMKII-mediated I(Ca) facilitation, demonstrating that phosphorylation of beta(2a) at Thr498 modulates native calcium channels. These findings reveal a molecular mechanism for targeting CaMKII to LTCCs and facilitating I(Ca) that may modulate Ca(2+) entry in diverse cell types coexpressing CaMKII and the beta(2a) subunit.     

Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

Microglia cells are the brain counterpart of macrophages and function as the first defense in the brain. Although they are neuroprotective in the young brain, microglia cells may be primed to react abnormally to stimuli in the aged brain and to become neurotoxic and destructive during neurodegeneration. Aging-induced immune senescence occurs in the brain as age-associated microglia senescence, which renders microglia to function abnormally and may eventually promote neurodegeneration. Microglia senescence is manifested by both morphological changes and alterations in immunophenotypic expression and inflammatory profile. These changes are likely caused by microinvironmental factors, but intrinsic factors cannot yet be completely excluded. Microglia senescence appears to underlie the switching of microglia from neuroprotective in the young brain to neurotoxic in the aged brain. The hypothesis of microglia senescence during aging offers a novel perspective on their roles in aging-related neurodegeneration. In Parkinson's disease and Alzheimer's disease, over-activation of microglia may play an active role in the pathogenesis because microglia senescence primes them to be neurotoxic during the development of the diseases.     

The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit

Sodium channels consist of a pore-forming alpha subunit and auxiliary beta 1 and beta 2 subunits. The subunit beta 1 alters the kinetics and voltage-dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N-terminal extracellular domain of beta 1, but does not require the intracellular C-terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain Na(V)1.2a sodium channel alpha subunits expressed in Chinese hamster lung cells by a mutant beta1 construct with 34 amino acids deleted from the C-terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co-immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant beta 1 with alpha, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10,000 times more mutant beta 1 RNA was required to achieve the level of functional modulation observed with injection of full-length beta 1. Together, these findings suggest that the C-terminal cytoplasmic domain of beta 1 is an important determinant of beta1 binding to the sodium channel alpha subunit in both mammalian cells and Xenopus oocytes.     

Structure of a voltage-dependent K+ channel beta subunit.

The integral membrane subunits of many voltage-dependent potassium channels are associated with an additional protein known as the beta subunit. One function of beta subunits is to modify K+ channel gating. We have determined the structure of the conserved core of mammalian beta subunits by X-ray crystallography at 2.8 A resolution. Like the integral membrane component of K+ channels, beta subunits form a four-fold symmetric structure. Each subunit is an oxidoreductase enzyme complete with a nicotinamide co-factor in its active site. Several structural features of the enzyme active site, including its location with respect to the four-fold axis, imply that it may interact directly or indirectly with the K+ channel's voltage sensor. This structure suggests a mechanism for coupling membrane electrical excitability directly to chemistry of the cell.     

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca²⁺-activated K⁺ channel (BK_Ca, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BK_Ca channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BK_Ca channel. Calculation of an electrostatic surface map of the BK_Ca gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BK_Ca channel activation gating.     

Involvement of the calcium channel beta3 subunit in olfactory signal transduction

Despite the expression of voltage-dependent Ca2+ channels in nasal turbinate epithelium, their role in odorant chemosensation has remained obscure. Therefore, we investigated olfactory neurotransduction in beta3-deficient mice. RT-PCR and Western blots confirmed the expression of various types of Ca2+ channels in the nasal turbinate. Electrophysiological evaluations revealed that beta3-null mice had a 60% reduction in the high-voltage-dependent Ca2+ currents in olfactory receptor neurons due to reduced N- and L-type channel currents. The beta3-null mice showed increased olfactory neuronal activity to triethylamine, and this effect was mimicked by the perfusion of the specific N-type Ca2+ channel inhibitor omega-conotoxin GVIA in the electro-olfactogram. Diluted male urine odors induced higher Fos immunoreactivity in the main olfactory bulbs of beta3-deficient mice, indicating enhanced signal transduction of odor information in these mice. Our data indicate the involvement of voltage-dependent Ca2+ channels and importance of the beta3 subunit in olfactory signal transduction.     

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca²⁺-activated K⁺ channel (BK_Ca, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BK_Ca channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BK_Ca channel. Calculation of an electrostatic surface map of the BK_Ca gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BK_Ca channel activation gating.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

The I-II loop of the Ca2+ channel alpha1 subunit contains an endoplasmic reticulum retention signal antagonized by the beta subunit

The auxiliary beta subunit is essential for functional expression of high voltage-activated Ca2+ channels. This effect is partly mediated by a facilitation of the intracellular trafficking of alpha1 subunit toward the plasma membrane. Here, we demonstrate that the I-II loop of the alpha1 subunit contains an endoplasmic reticulum (ER) retention signal that severely restricts the plasma membrane incorporation of alpha1 subunit. Coimmunolabeling reveals that the I-II loop restricts expression of a chimera CD8-I-II protein to the ER. The beta subunit reverses the inhibition imposed by the retention signal. Extensive deletion of this retention signal in full-length alpha1 subunit facilitates the cell surface expression of the channel in the absence of beta subunit. Our data suggest that the beta subunit favors Ca2+ channel plasma membrane expression by inhibiting an expression brake contained in beta-binding alpha1 sequences.     

The beta subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca2+ channel.

The skeletal muscle (SKM) L-type Ca2+ channel is composed of a central subunit designated alpha 1, which contains the pore and the dihydropyridine (DHP) binding domains and three associated subunits, alpha 2/delta, beta, and gamma, which influence the activity of the SKM alpha 1. Coexpression of SKM alpha 1 and SKM beta in stably transfected mouse L cells results in a dramatic increase in DHP binding accompanied by fast gated Ba2+ currents. We report here that this "SKM alpha 1 beta-related phenotype" can be converted upon intracellular trypsin treatment into a slowly inactivating, DHP sensitive "SKM alpha 1 phenotype." These observations indicate that current amplitude, fast inactivation, and DHP sensitivity are modulated by an interaction of SKM alpha 1 and SKM beta on the internal side of the membrane.     

Structure of the calcium channel beta subunit: the place of the beta-interaction domain

Voltage-gated calcium channels are key players in a number of fundamental physiological functions including contraction, secretion, transmitter release or gene activation. They allow a flux of calcium into the cell that constitutes a switch-on signal for most of these functions. The structures responsible for the shaping of these fluxes by the membrane voltage belong to the channel itself, but a number of associated proteins are known to more precisely tune this calcium entry and adapt it to the cellular demand. The calcium channel regulatory beta subunit is undoubtedly the most important one, being influent on the expression, the kinetics, the voltage-dependence of channel opening and closing and on the pharmacology of the channel. Heterologous expression, combined to mutagenesis and electrophysiological and biochemical experiments have revealed the roles of short sequences of the beta subunit, including the BID (beta-interaction domain), in the physical and functional interactions with the channel pore. The resolved crystal structure of the beta subunit now sheds new light on these sequences and their interactions with the rest of the protein. The presence of a type 3 src-homology (SH3) domain and a guanylate kinase (GK) domain confirms that the subunit belongs to the MAGUK protein family. Consistently, the polyproline binding site and the kinase function of the SH3 and the GK domains, respectively, are non functional, and the BID appears to be buried in the structure, preserving the SH3-GK interaction but not directly available for interactions with the channel pore subunit. Anchoring of the beta subunit to the channel occurs via a hydrophobic grove in the GK domain, leaving a large surface of the subunit open to other protein-protein interactions. To what extent the intramolecular SH3-GK interaction is necessary for the stabilisation of this grove in a functional unit remains to be understood. The beta subunit may thus play a key role in scaffolding multiple proteins around the channel and organizing diverse calcium-dependent signalling pathways directly linked to voltage-gated calcium entry. These findings will undoubtedly vitalize the search for new beta-specific partners and functions.     

The GK domain of the voltage-dependent calcium channel beta subunit is essential for binding to the alpha subunit

The β subunits of voltage-dependent calcium channels bind the pore-forming α₁ subunit and play an important role in the regulation of calcium channel function. Recently, we have identified a new splice variant of the β₄ subunit, which we have termed the β_4d subunit. The β_4d subunit is a truncated splice variant of the β_4b subunit and lacks parts of the guanylate kinase (GK) domain and the C-terminus. The calcium current in BHK cells expressing α_1C and α₂δ with the β_4d subunit was as small as that without the β_4d subunit. Western blot analysis revealed that β_4d protein was expressed to a lesser extent that the β_4b protein. In addition, a GST pull down assay showed that the β_4d subunit could not interact with the α₁ subunit of the calcium channel. Collectively, our results suggest that the GK domain of the β subunit is essential for the expression of the functional calcium channel.