Edited by Mike Shipston The cardiac mechanosensitive BK (Slo1) channels are gated by Ca 2؉ , voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (P o) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (O) and shortening the closed-time constant (C). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.
Background and Purpose: The Slo3 potassium (K Ca 5.1) channel, which is specifically expressed in the testis and sperm, is essential for mammalian male fertilization. The sequence divergence of the bovine, mouse and human Slo3 α-subunit revealed a rapid evolution rate across different species. The rat Slo3 (rSlo3) channel has not been cloned and characterized previously. Experimental Approach: We used molecular cloning, electrophysiology (inside-out patches and outside-out patches) and mutagenesis to investigate the biophysical properties and pharmacological characteristics of the rSlo3 channel.
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+.
binding to the EGF receptor (EGFR), triggers tyrosine kinase-dependent signaling which effectively down-regulates Kv1.3 currents. However, EGF induces internalization of many membrane proteins, but no evidence linked a decrease of Kv1.3 current amplitude as a consequence of channel endocytosis. Here we describe that Kv1.3 undergoes EGF-dependent endocytosis. Kv1.3 redistribution upon EGFR activation corresponds to induced-endocytosed channels which colocalized intracellular Kv1.3 with endocytic vesicles markers. Our studies revealed that tyrosines and other putative motive sequences are not apparently important for EGFR activation-mediated internalization. However, a putative sequence for ERK1/2-mediated threonine phosphorylation revealed its potential importance in surface Kv1.3 down-regulation. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited EGFR activation-mediated internalization of Kv1.3. We demonstrated that changes in Kv1.3 distribution upon EGFR activation corresponded to a massive Kv1.3 endocytosis via clathrin-coated pits mechanisms which localized the Kv1.3 channels in the lysosomal degradative pathway. This EGF mechanism is highly relevant because halted proliferation but increased neuronal migration from the subventricular zone of the forebrain to the olfactory bulb. Supported by MINECO, Spain (BFU2014-54928-R, BFU2015-70067-REDC and FEDER).
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