Divalent regulation and intersubunit interactions of human Connexin26 (Cx26) hemichannels

Abstract

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca(2+) and Mg(2+) of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca (2+). To effect hemichannel closure, the apparent affinity of Ca(2+) (0.33 mM) is higher than for Mg(2+) (1.8 mM). Hemichannel closure is accelerated by physiological Ca(2+) concentrations, but non-physiological concentrations of extracellular Mg(2+) are required for this effect. Our recent report provided evidence that extracellular Ca(2+) facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca(2+) compared with the D50-K61 interaction.

Mechanism of gating by calcium in connexin hemichannels

“…Only a few point mutations led to functional hemichannels (D46C, E47Q, D50N, R184K), but all showed accelerated deactivation kinetics at all extracellular Ca 2+ concentrations. We previously showed that Cx26 hemichannels deactivate very slowly at low extracellular Ca 2+ because the electrostatic interaction between D50 and K61 is less likely to be disrupted, which stabilizes the hemichannel in the open state (17,19). Charge-changing mutations of these residues that weaken the interaction accelerate the closing kinetics (17,19), similar to results seen with D46C, E47Q, and R184K mutant hemichannels.…”

“…We previously showed that Cx26 hemichannels deactivate very slowly at low extracellular Ca 2+ because the electrostatic interaction between D50 and K61 is less likely to be disrupted, which stabilizes the hemichannel in the open state (17,19). Charge-changing mutations of these residues that weaken the interaction accelerate the closing kinetics (17,19), similar to results seen with D46C, E47Q, and R184K mutant hemichannels. In addition, in steady-state measurements, mutations of D50 and E47 significantly reduce the apparent affinity for Ca 2+ , suggesting that they also reduce the ability of Ca 2+ to stabilize the closed state.…”

“…Recently, we found that extracellular Ca 2+ destabilizes the open state of hemichannels, at least in human Cx26 (hCx26) and hCx30, by disrupting a salt-bridge interaction between residues D50 and K61 located close to the extracellular entrance of the pore (17,19). This open-state destabilization facilitates hemichannel closure (17,19).…”

“…This open-state destabilization facilitates hemichannel closure (17,19). However, disruption of this interaction does not seem to be a general mechanism for Ca 2+ regulation of connexins, because these charges at these positions are not highly conserved across the family of connexin proteins.…”

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

“…Xenopus oocytes were injected with the same amount of Cx45 WT or 6E cRNA, together with DNA antisense to the endogenous XenCx38. The oocytes were voltage-clamped and macroscopic currents of the hemichannels recorded during steps to depolarizing voltages to activate the channels [44, 45] (Fig. 8).…”