Two classes of regulatory subunits coassemble in the same BK channel and independently regulate gating.
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ABSTRACT:
SUBMITTER: Gonzalez-Perez V
PROVIDER: S-EPMC4578311 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Calcium- and voltage-activated potassium channels (BK) are regulated by a multiplicity of signals. The prevailing view is that different BK gating mechanisms converge to determine channel opening and that these gating mechanisms are allosterically coupled. In most instances the pore forming ? subunit of BK is associated with one of four alternative ? subunits that appear to target specific gating mechanisms to regulate the channel activity. In particular, ?1 stabilizes the active configuration of the BK voltage sensor having a large effect on BK Ca(2+) sensitivity. To determine the extent to which ? subunits regulate the BK voltage sensor, we measured gating currents induced by the pore-forming BK ? subunit alone and with the different ? subunits expressed in Xenopus oocytes (?1, ?2IR, ?3b, and ?4). We found that ?1, ?2, and ?4 stabilize the BK voltage sensor in the active conformation. ?3 has no effect on voltage sensor equilibrium. In addition, ?4 decreases the apparent number of charges per voltage sensor. The decrease in the charge associated with the voltage sensor in ? ?4 channels explains most of their biophysical properties. For channels composed of the ? subunit alone, gating charge increases slowly with pulse duration as expected if a significant fraction of this charge develops with a time course comparable to that of K(+) current activation. In the presence of ?1, ?2, and ?4 this slow component develops in advance of and much more rapidly than ion current activation, suggesting that BK channel opening proceeds in two steps.
| S-EPMC3503226 | biostudies-literature
Project description:Most human genes contain multiple alternative splice sites believed to extend the complexity and diversity of the proteome. However, little is known about how interactions among alternative exons regulate protein function. We used the Caenorhabditis elegans slo-1 large-conductance calcium and voltage-activated potassium (BK) channel gene, which contains three alternative splice sites (A, B, and C) and encodes at least 12 splice variants, to investigate the functional consequences of alternative splicing. These splice sites enable the insertion of exons encoding part of the regulator of K(+) conductance (RCK)1 Ca(2+) coordination domain (exons A1 and A2) and portions of the RCK1-RCK2 linker (exons B0, B1, B2, C0, and C1). Exons A1 and A2 are used in a mutually exclusive manner and are 67% identical. The other exons can extend the RCK1-RCK2 linker by up to 41 residues. Electrophysiological recordings of all isoforms show that the A1 and A2 exons regulate activation kinetics and Ca(2+) sensitivity, but only if alternate exons are inserted at site B or C. Thus, RCK1 interacts with the RCK1-RCK2 linker, and the effect of exon variation on gating depends on the combination of alternate exons present in each isoform.
| S-EPMC3251094 | biostudies-literature
Project description:Large conductance K+ channels, termed BK channels, regulate a variety of cellular and physiological functions. Although universally activated by changes in voltage or [Ca2+ ]i , the threshold for BK channel activation varies among loci of expression, often arising from cell-specific regulatory subunits including a family of leucine rich repeat-containing (LRRC) γ subunits (LRRC26, LRRC52, LRRC55 and LRRC38). The 'founding' member of this family, LRRC26, was originally identified as a tumour suppressor in various cancers. An LRRC26 knockout (KO) mouse model recently revealed that LRRC26 is also highly expressed in secretory epithelial cells and partners with BK channels in the salivary gland and colonic goblet cells to promote sustained K+ fluxes likely essential for normal secretory function. To accomplish this, LRRC26 negatively shifts the range of BK channel activation such that channels contribute to K+ flux near typical epithelial cell resting conditions. In colon, the absence of LRRC26 increases vulnerability to colitis. LRRC26-containing BK channels are also likely important regulators of epithelial function in other loci, including airways, female reproductive tract and mammary gland. Based on an LRRC52 KO mouse model, LRRC52 regulation of large conductance K+ channels plays a role both in sperm function and in cochlear inner hair cells. Although our understanding of LRRC-containing BK channels remains rudimentary, KO mouse models may help define other organs in which LRRC-containing channels support normal function. A key topic for future work concerns identification of endogenous mechanisms, whether post-translational or via gene regulation, that may impact LRRC-dependent pathologies.
| S-EPMC8930516 | biostudies-literature
Project description:In most mammalian tissues, Ca2+i/voltage-gated, large conductance K+ (BK) channels consist of channel-forming slo1 and auxiliary (β1-β4) subunits. When Ca2+i (3-20 µM) reaches the vicinity of BK channels and increases their activity at physiological voltages, β1- and β4-containing BK channels are, respectively, inhibited and potentiated by intoxicating levels of ethanol (50 mM). Previous studies using different slo1s, lipid environments, and Ca2+i concentrations-all determinants of the BK response to ethanol-made it impossible to determine the specific contribution of β subunits to ethanol action on BK activity. Furthermore, these studies measured ethanol action on ionic current under a limited range of stimuli, rendering no information on the gating processes targeted by alcohol and their regulation by βs. Here, we used identical experimental conditions to obtain single-channel and macroscopic currents of the same slo1 channel ("cbv1" from rat cerebral artery myocytes) in the presence and absence of 50 mM ethanol. First, we assessed the role five different β subunits (1,2,2-IR, 3-variant d, and 4) in ethanol action on channel function. Thus, two phenotypes were identified: (1) ethanol potentiated cbv1-, cbv1+β3-, and cbv1+β4-mediated currents at low Ca2+i while inhibiting current at high Ca2+i, the potentiation-inhibition crossover occurring at 20 µM Ca2+i; (2) for cbv1+β1, cbv1+wt β2, and cbv1+β2-IR, this crossover was shifted to ∼3 µM Ca2+i Second, applying Horrigan-Aldrich gating analysis on both phenotypes, we show that ethanol fails to modify intrinsic gating and the voltage-dependent parameters under examination. For cbv1, however, ethanol (a) drastically increases the channel's apparent Ca2+ affinity (nine-times decrease in Kd) and (b) very mildly decreases allosteric coupling between Ca2+ binding and channel opening (C). The decreased Kd leads to increased channel activity. For cbv1+β1, ethanol (a) also decreases Kd, yet this decrease (two times) is much smaller than that of cbv1; (b) reduces C; and (c) decreases coupling between Ca2+ binding and voltage sensing (parameter E). Decreased allosteric coupling leads to diminished BK activity. Thus, we have identified critical gating modifications that lead to the differential actions of ethanol on slo1 with and without different β subunits.
| S-EPMC5089933 | biostudies-literature
Project description:The properties and physiological function of pore-forming ?-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are potently modified by their functional coupling with regulatory subunits in many tissues. However, mechanisms that might control functional coupling are very poorly understood. Here we show that S-acylation, a dynamic post-translational lipid modification of proteins, of the intracellular S0-S1 loop of the BK channel pore-forming ?-subunit controls functional coupling to regulatory ?1-subunits. In HEK293 cells, ?-subunits that cannot be S-acylated show attenuated cell surface expression, but expression was restored by co-expression with the ?1-subunit. However, we also found that nonacylation of the S0-S1 loop reduces functional coupling between ?- and ?1-subunits by attenuating the ?1-subunit-induced left shift in the voltage for half-maximal activation. In mouse vascular smooth muscle cells expressing both ?- and ?1-subunits, BK channel ?-subunits were endogenously S-acylated. We further noted that S-acylation is significantly reduced in mice with a genetic deletion of the palmitoyl acyltransferase (Zdhhc23) that controls S-acylation of the S0-S1 loop. Genetic deletion of Zdhhc23 or broad-spectrum pharmacological inhibition of S-acylation attenuated endogenous BK channel currents independently of changes in cell surface expression of the ?-subunit. We conclude that functional effects of S-acylation on BK channels depend on the presence of ?1-subunits. In the absence of ?1-subunits, S-acylation promotes cell surface expression, whereas in its presence, S-acylation controls functional coupling. S-Acylation thus provides a mechanism that dynamically regulates the functional coupling with ?1-subunits, enabling an additional level of conditional, cell-specific control of ion-channel physiology.
| S-EPMC6690687 | biostudies-literature
Project description:Calcium ions bind at the gating ring which triggers the gating of BK channels. However, the allosteric mechanism by which Ca2+ regulates the gating of BK channels remains obscure. Here, we applied Molecular Dynamics (MD) and Targeted MD to the integrated gating ring of BK channels, and achieved the transition from the closed state to a half-open state. Our date show that the distances of the diagonal subunits increase from 41.0 Å at closed state to 45.7Å or 46.4 Å at a half-open state. It is the rotatory motion and flower-opening like motion of the gating rings which are thought to pull the bundle crossing gate to open ultimately. Compared with the 'Ca2+ bowl' at RCK2, the RCK1 Ca2+ sites make more contribution to opening the channel. The allosteric motions of the gating ring are regulated by three group of interactions. The first weakened group is thought to stabilize the close state; the second strengthened group is thought to stabilize the open state; the third group thought to lead AC region forming the CTD pore to coordinated motion, which exquisitely regulates the conformational changes during the opening of BK channels by Ca2+.
| S-EPMC5617150 | biostudies-literature
Project description:Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, ? and ?, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four ? subunits per channel, with each ? subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a ?1 subunit (or single type of ?1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of ?1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four ?1 subunits, but a single ?1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+ channel.
| S-EPMC6176617 | biostudies-literature
Project description:Large conductance, voltage- and Ca2+-activated K+ (BK(Ca)) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four alpha subunits of BK(Ca) may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BK(Ca) gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BK(Ca) activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BK(Ca) channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.
| S-EPMC2266574 | biostudies-literature
Project description: Not available
| S-EPMC3149440 | biostudies-literature
Project description:High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. beta1 and beta2 subunits increase apparent channel calcium sensitivity. The beta1 subunit also decreases the voltage sensitivity of the channel and the beta2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the beta1 and beta2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a beta2 subunit without its N-type inactivation domain (beta2IR). The results indicate that the beta2IR subunit, like the beta1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the beta1 subunit, the beta2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied beta subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the beta1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both beta subunits reduce the intrinsic equilibrium constant for channel opening (L0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the beta1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the alpha subunit is coexpressed with the beta2IR subunit.
| S-EPMC2217511 | biostudies-literature