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

Julian Thimm,Adam Mechler,Hai Lin,Seung Rhee,Ratnesh Lal

doi:10.1074/jbc.m412749200

Abstract

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 approximately 1.8 nm, consistent with the closed channel conformation. Reducing Ca(+2) concentration to approximately 1.4, 1, and 0 mm, which changes hemichannels from the closed to open conformation, increased the pore diameter to approximately 2.5 nm for approximately 27, 74, and 100% of hemichannels, respectively. Thus, open/close probability of the hemichannel appears to be [Ca(2+)]-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.

Highlights

Gap junctions are composed of two hemichannels in the plasma membranes of apposing cells
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
Six subunits representing constituent connexins are visible in many connexons, and there is an unambiguous presence of a pore with an indentation depth up to ϳ0.8 nm when imaged in nominal calcium-free buffer

Summary

Introduction

Gap junctions are composed of two hemichannels (or connexons) in the plasma membranes of apposing cells They allow the diffusion-driven transfer of ions and small cytoplasmic molecules between interconnected cells, synchronize electrical activity, and regulate metabolic homeostasis, cell growth, and differentiation Unlike intercellular gap junctional channels (exposed to only intracellular ionic milieu), activity of non-junctional hemichannels (exposed to both intraand extracellular milieux) appears to be modulated in a manner similar to other conventional ion channels (for a review, see Ref. 25). AFM1 images of Ca2ϩ-dependent conformational change on the cytoplasmic side of Cx26 connexons present as crystalline patches in the gap junctions and on the extracellular side exposed after the force dissection of the gap junctions are available [9]. We further used AFM phase mode imaging and computational analysis [31] for the first time on membrane channels to reveal that extracellular hydrophobic domains of connexins regulate calcium-dependent conformational changes

Methods

Results

Conclusion