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J. Biol. Chem., Vol. 280, Issue 11, 10646-10654, March 18, 2005

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Calcium-dependent Open/Closed Conformations and Interfacial Energy Maps of Reconstituted Hemichannels^*

Julian Thimm, Adam Mechler¶, Hai Lin, Seung Rhee||**, and Ratnesh Lal

From the Neuroscience Research Institute, University of California, Santa Barbara, California, 93106 and the ^||Department of Biochemistry, Yeungnam University, Kyoungsan, 712-749, Republic of Korea

Using an atomic force microscope, we have studied three-dimensional molecular topography and calcium-sensitive conformational changes of individual hemichannels. Full-length (non-truncated) Cx43 hemichannels (connexons), when reconstituted in lipid bilayer, appear as randomly distributed individual particles and clusters. They show a lack of preferential orientation of insertion into lipid membrane; in a single bilayer, connexons with protrusion of either the extracellular face or the large non-truncated cytoplasmic face are observed. Extracellular domains of these undocked hemichannels are structurally different from hemichannels in the docked gap junctional plaques examined after their exposure by force dissection or chemical dissection. Calcium induced a reversible change in the extracellular pore diameter. Hemichannels imaged in a physiological buffer with 1.8 mM Ca⁺² had the pore diameter of 1.8 nm, consistent with the closed channel conformation. Reducing Ca⁺² concentration to 1.4, 1, and 0 mM, which changes hemichannels from the closed to open conformation, increased the pore diameter to 2.5 nm for 27, 74, and 100% of hemichannels, respectively. Thus, open/close probability of the hemichannel appears to be [Ca²⁺]-dependent. Computational analysis of the atomic force microscopy phase mode imaging reveals a significantly higher interfacial energy for open hemichannels that results from the interactions between the atomic force microscope probe and the hydrophobic domains. Thus, hydrophobic extracellular domains of connexins regulate calcium-dependent conformational changes.

Received for publication, November 11, 2004 , and in revised form, December 21, 2004.

^* This work was supported in part by a joint award from National Institute of General Medical Sciences and National Institute of Aging (GM056290) and the Philip Morris External Grant Program. Part of this work has been presented in an abstract form in 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^¶ Partly supported by a NATO-NSF Postdoctoral Fellowship in Science and Engineering grant.

^** Supported by the Korean Ministry of Education (Grant GE 97-124).

To whom correspondence should be addressed. Tel.: 805-893-2350; Fax: 805-893-2005; E-mail: lal{at}lifesci.ucsb.edu.

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