Molecular basis of calcium regulation in connexin-32 hemichannels.
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Journal: 2004/April - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca2+ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca2+ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of approximately 90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels ( approximately 18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca2+ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca2+ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca2+ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.
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Proc Natl Acad Sci U S A 100(26): 16030-16035
PMID: 14663144

Molecular basis of calcium regulation in connexin-32 hemichannels

Unit of Experimental Neurology, Research Department, “Ramón y Cajal” Hospital, Carretera de Colmenar Viejo km 9, 28034 Madrid, Spain
To whom correspondence should be addressed. E-mail: se.crh@oirrab.c.siul.
J.M.G.-H. and M.d.M. contributed equally to this work.
Edited by Michael V. L. Bennett, Albert Einstein College of Medicine, Bronx, NY
Edited by Michael V. L. Bennett, Albert Einstein College of Medicine, Bronx, NY
Received 2003 Jan 20; Accepted 2003 Oct 13.
Copyright © 2003, The National Academy of Sciences

Abstract

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of ≈90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels (≈18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.

Abstract

Vertebrate gap-junction channels are made up of connexins (Cxs), a gene family encoding at least 20 different isoforms (1). Each gap-junction channel is built up by the docking of two hemichannels, one from each of the neighboring cells. In addition to contributing to gap-junction channels, a subset of Cxs make open hemichannels in the plasma membrane that are capable of fulfilling a role other than that of gap-junction-mediated cell-to-cell communication. Cxs oligomerize into hexameric structures, i.e., hemichannels, before reaching the cell surface (2), and thus the presence of undocked hemichannels in the plasma membrane constitutes a normal phase in the life cycle of gap-junction proteins. Although it was thought that hemichannels remained closed in the plasma membrane until a newly formed channel acquires an open configuration, analysis of rat Cx46 in Xenopus oocytes revealed that the hemichannels can be voltage-gated by depolarization in the nonjunctional plasma membrane under physiological conditions (3). It is now known that this capacity to form functional hemichannels is widely extended among members of the Cx family, and that functional hemichannels are present in many native cells (4–7).

Hemichannels permit the rapid exchange of ions and of small molecules between the cytoplasm and the extracellular space (8–12). As such, they have been implicated in the regulation of various physiological processes (11, 13–15), as well as in the pathogenesis of certain disorders (16–19). The activation of hemichannels depends critically on the external Ca concentration ([Ca]o). External Ca ions are known to affect the voltage sensitivity of gating (20, 21) and can induce reversible conformational changes of hemichannel structure, compatible with a mechanism of gating (22). However, the molecular basis for this regulation remains unknown. We show here that the direct interaction of divalent cations with a site in the external vestibule of the pore mediates most Ca effects on the Cx32 hemichannels. This binding site is responsible for preventing voltage-gated opening of hemichannels to the higher conductance sublevel (90 pS) and also for blocking inward currents through the lower conductance open state (18 pS). Thus, divalent cation blockage is a prominent feature in the physiology of Cx32 hemichannels. We also report that in a Cx32 mutant associated with a hereditary peripheral neuropathy, this binding site is destroyed, causing the complete Ca deregulation of these hemichannels.

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Acknowledgments

We thank Gina Sosinsky and Juan Lerma for helpful discussions and Rosa Barquero for technical assistance. This work was supported by the European Commission (Grant QLG1-1999/00516 to L.C.B.), the Ministerio de Ciencia y Tecnología (Grant SAF2001-0048 to L.C.B.), and the Comunidad de Madrid (Grant 08.5/0069/2001 to L.C.B.). J.M.G.-H. is a “Ramón y Cajal” Program researcher.

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Cx, connexin; [Ca2+]o, external Ca2+ concentration; CMTX, X-linked Charcot–Marie–Tooth disease; SIS, standard internal solution.

Notes
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Cx, connexin; [Ca2+]o, external Ca2+ concentration; CMTX, X-linked Charcot–Marie–Tooth disease; SIS, standard internal solution.

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