Extracellular Divalent Cations Regulation of Cx26 and Cx30 Hemichannels

Abstract

Due to the large size and modest selectivity of the aqueous pore, exacerbated opening of connexin hemichannels leads to loss of electrochemical gradients and of small cytoplasmic metabolites, causing cell death. Control of hemichannel opening is indispensable, and is achieved by extracellular divalent concentrations, which drastically reduces hemichannel activity. Here, we explore the differences between extracellular Ca2+ and Mg2+ regulation in two relatives connexin, hCx26 and hCx30. Our standard protocol for assessment connexin hemichannel activation and deactivation with the two electro-voltage clamp technique is to examine the peak tail currents and their relaxation kinetics following a depolarizing pulse from −80 mV to 0 mV. using this protocol, the peak tail currents increase with reduction of external divalents. We estimate the extracellular Ca2+ and Mg2+ apparent affinity for hCx26 hemichannels at values of 0.33 mM and 1.8 mM, respectively. hCx30 hemichannels showed slightly higher extracellular Ca2+ and Mg2+ apparent affinity with values of 0.17 mM and 1.0 mM, respectively. At physiological Ca2+ concentration (1.0 - 1.8 mM), both hCx26 and hCx30 hemichannels reach ≤ 15% of the maximal response, but at corresponding Mg2+ concentrations they reach ≥ 50%. In addition, deactivation time constant at the tail currents are accelerated as a function of Ca2+ concentrations in hCx26 and hCx30 hemichannels; however, only high extracellular Mg2+ concentrations (> 2.0 mM) are capable to accelerate deactivation kinetics in both connexin types. The holding currents at steady state are significantly increased at physiological extracellular Mg2+ concentrations (1.0 - 1.2 mM) in both hCx26 and hCx30 suggesting an increase in open hemichannels even at negative potentials. Our data support that, under physiological ionic conditions, Ca2+, but not Mg2+ plays a major role stabilizing and facilitating closing of Cx26 and Cx30 hemichannels. Support: R01GM099490