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Modulation of BK channel gating by the ß2 subunit involves both membrane-spanning and cytoplasmic domains of Slo1.

ABSTRACT: Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory β subunits. Four types of BK channel β subunits (β1-β4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the β1 and β2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these β subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for β2 modulation based on the study of differential effects of the β2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (βA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the β2 but not for the β1 modulation. These results suggest that the β2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel β subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.

SUBMITTER: Lee US

PROVIDER: S-EPMC3073600 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices"). Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right-handed bundle structures along a common 4-fold axis.

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Modulation of BK channel gating by the ß2 subunit involves both membrane-spanning and cytoplasmic domains of Slo1.

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