Abstract
The opening of connexin (Cx) hemichannels in the membrane is tightly regulated by calcium (Ca2+) and membrane voltage. Electrophysiological and atomic force microscopy experiments indicate that Ca2+ stabilizes the hemichannel closed state. However, structural data show that Ca2+ binding induces an electrostatic seal preventing ion transport without significant structural rearrangements. In agreement with the closed-state stabilization hypothesis, we found that the apparent Ca2+ sensitivity is increased as the voltage is made more negative. Moreover, the voltage and Ca2+ dependence of the channel kinetics indicate that the voltage sensor movement and Ca2+ binding are allosterically coupled. An allosteric kinetic model in which the Ca2+ decreases the energy necessary to deactivate the voltage sensor reproduces the effects of Ca2+ and voltage in Cx46 hemichannels. In agreement with the model and suggesting a conformational change that narrows the pore, Ca2+ inhibits the water flux through Cx hemichannels. We conclude that Ca2+ and voltage act allosterically to stabilize the closed conformation of Cx46 hemichannels.
Highlights
Connexins (Cxs) are transmembrane proteins that can form two types of non-selective channels, gap junction channels and hemichannels
Verselis and Srinivas found that Cx46 hemichannels close even in absence of divalent cations, ruling out the possibility of a divalent block as the gating mechanism, and proposed that external divalent cations stabilize the slow gate closures induced by hyperpolarization[18]
Can Ca2+ bind to the open state? Is Ca2+ binding voltage dependent? Can Ca2+ affect the ion flux through an electrostatic mechanism? What is the mechanism through which Ca2+ is stabilizing the closed states? To address these issues we tested the effects of voltage and Ca2+ in Cx46 hemichannels expressed in X. laevis oocytes
Summary
Connexins (Cxs) are transmembrane proteins that can form two types of non-selective channels, gap junction channels and hemichannels. AFM imaging of Cx hemichannels shows that in the presence of Ca2+ the extracellular mouth of the channel narrows[17,18,21] Taken together, these results support the hypothesis that voltage-dependent gating is an intrinsic property of the slow gate and divalent cations stabilize the closed state(s). Further analysis of the channel kinetics reveals two distinguishable kinetic steps and these are directly regulated by voltage, with Ca2+ accelerating the deactivation kinetics which reaches a constant value at high Ca2+ concentrations. This cannot be explained by a linear mechanism and indicate that Ca2+ binding is allosterically coupled to the voltage sensor. A detailed analysis of the activation and deactivation channel kinetics suggests that Ca2+ and voltage sensors are allosterically coupled to promote Cx46 hemichannel closing
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