A Human Disease Mutation (D50N) Reveals Insights on the Mechanisms for Ca2+ Regulation in Human Connexin26 (hCX26) Hemichannels

Abstract

Connexin hemichannels allow the release of small metabolites, such as ATP and glutamate, which play important roles in autocrine/paracrine signaling in a variety of cell types. Exacerbated hemichannel opening, however, leads to loss of electrochemical gradients and metabolites, causing cell death. Control of hemichannel opening is indispensable, and is achieved by physiological extracellular Ca2+, which drastically reduces hemichannel activity. Here, we explore the molecular mechanism of Ca2+ regulation in hCx26 hemichannels. We found that outward and tail currents are greatly increased with reduction of external Ca2+. The data show that deactivation kinetics are accelerated as a function of Ca2+ concentration suggesting that Ca2+ facilitates closing of the channels. In addition, we found that an Asp to Asn mutation at position 50 (D50N) - a human mutation that causes disease - has lower apparent affinity for Ca2+ compared with wild-type. Strikingly, unlike wild-type channels, the deactivation kinetics of D50N channels were unaffected by changes in Ca2+. To explore the role of Ca2+ interactions with a negatively charged residue at position 50, we substituted this position by a cysteine residue (D50C) and performed chemical modification with positively or negatively charged methanethiosulfonate (MTS) reagents. The D50C mutant hemichannels display similar properties to those of D50N mutants. Chemical modification of D50C with a negatively charged reagent, MTSES, causes the channels to largely return to wild-type sensitivity to Ca2+. Conversely, positively charged MTS reagents have no effect. In the crystal structure of hCx26 channels, position D50 faces the pore lumen and forms an inter-subunit salt bridge interaction with position K61. Because this interaction appears to take place in open conformation, we propose that Ca2+ facilitates the closing of hCx26 hemichannels by disrupting this salt bridge.