Heterotypic docking of Cx43 and Cx45 connexons blocks fast voltage gating of Cx43

ABSTRACT Immunohistochemical co-localization of distinct connexins (Cxs) in functional areas suggests the formation of heteromultimeric channels. To determine the docking effects of the heterotypic combination of Cx43 and Cx45 on the voltage-gating properties of their channels, we transfected DNA encoding Cx43 or Cx45 into N2A neuroblastoma or HeLa cells. Using a double whole-cell voltage-clamp technique, we determined macroscopic and single-channel gating properties of the intercellular channels formed. Cx43-Cx45 heterotypic channels had rectifying properties where Cx45 connexons inactivated rapidly upon hyperpolarizing voltage pulses applied to the Cx45-expressing cell. During depolarizing pulses to the Cx45-expressing cell, Cx43 connexons inactivated with substantially reduced kinetics as compared with homotypic Cx43 channels. Similar slow kinetics was observed for homotypic Cx43M257 (truncation mutant). Heterotypic channels had a main conductance whose value was predicted by the sum of corresponding homomeric connexon conductances; it was not voltage dependent and had no detectable residual conductance. The voltage-gating kinetics of heterotypic channels and their single-channel behavior implicate a role for the Cx43 carboxyl-terminal domain in the fast gating mechanism and in the establishment of residual conductance. Our results also suggest that heterotypic docking may lead to conformational changes that inhibit this action of the Cx43 carboxyl-terminal domain.

INTRODUCTION

Gap junctions contain intercellular channels that allow communication between adjacent cells. Connexins constitute a homologous family of gap junction proteins. A connexon (or hemichannel) is formed by the oligomerization of six connexin subunits, and the assembly and docking of two connexons leads to the formation of a complete gap junction channel.

Multiple connexins are expressed in the mammalian heart and many other organs (Kanter et al., 1993; Gros and Jongsma, 1996; Coppen et al., 1998). Using specific antibodies, connexin43 (Cx43), connexin40 (Cx4O), and connexin45 (Cx45) have been detected in the working myocardium. Other connexins have also been described in this organ, but their expression either is restricted to endothelial cells (Cx37) (Reed et al., 1993; Haefliger et al., 2000) or only their mRNA has been identified in the heart (Cx46) (Paul et al., 1991).

The co-existence of connexins in tissue indicates that channels could be assembled with more than one type of connexin. A complete new nomenclature for the different possible configurations has already been generated (Wang and Peracchia, 1998). The most relevant issue for channels formed of different connexins is that each one could provide the channels with different gating and permeability properties. Therefore, the interactions between these isoforms could result in channels with highly complex gating mechanisms. To fully understand the outcome of these interactions, it has been necessary to use simple cellular systems in which the expression and assembly of hemichannels among different cells can be controlled. One of the simplest configurations where. connexin interaction can be studied is the heterotypic channel, which results from the assembly of two different homomeric hemichannels (Fig. 1).

In this manuscript, we have further characterized the effects of heterotypic docking between Cx43 and Cx45 on gating produced by transjunctional voltage. Despite the homology of connexin sequences, strong differences exist in their gating and permeability properties. The channels formed by these connexins are sensitive to transjunctional voltage, as shown in studies performed in cellular systems that permit the expression of exogenous genes, such as Xenopus oocytes (Werner et al., 1989; Steiner and Ebihara, 1996) or transfected cells (Moreno et al., 1995a,b). The use of transfected cells has several advantages over Xenopus oocyte expression, including possible differences in behavior of mammalian connexins expressed in non-mammalian cells. Besides, transfection of cDNA into communication-- deficient tumor cell lines has become a standard procedure (Moreno et al., 1991) that allows single-channel recordings, necessary to elaborate a complete gating model of these mammalian connexins.

For our studies, we stably transfected cloned cDNAs for rat Cx43 (rCx43) (Beyer et al., 1987), chicken Cx45 (chCx45) (Beyer, 1990), or mouse Cx45 (mCx45) (Hennemann et al., 1992) in two different tumor cell lines: HeLa and neuroblastoma N2A. Having ascertained the individual gating behavior and unitary conductances for the channels containing only Cx43 or Cx45, we then performed studies on cell pairs that were forced to form heterotypic channels. To determine whether opposite gating polarities of Cx43 and Cx45 were responsible for the observed behaviors, we performed studies on heterotypic channels formed by wild-type Cx43 and Cx43M257, a mutant of Cx43 that is insensitive to pH gating (EkVitorin et al., 1996) and lacks the fast component of voltage-dependent inactivation (Revilla et al., 1999).

Our data provide evidence that the heterotypic combination of homomeric Cx43 and Cx45 connexons generates channels with complex behavior, where the voltage-gating mechanism of Cx43 becomes impaired after docking, and the residual conductance of the channels is no longer detectable. The data also imply that connexon interaction participates in the modulation of intercellular communication.

MATERIALS AND METHODS

Cells in culture and transfection

We thank Dr. Steve Taffet for allowing us to use the Cx43M257 transfected cells. We thank also Patricia L. Mantel for her technical and writing assistance.

This work was supported by National Institutes of Health grants 1IL63969 and HL50485 to A.P.M. and HL59199 and HDO9402 to E.C.B.

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[Author Affiliation]

Sergio Elenes,* Agustin D. Martinez,^ Mario Delmar,^^ Eric C. Beyer,^ and Alonso P. Moreno*

[Author Affiliation]

*Krannert Institute of Cardiology, Indiana University, Indianapolis, Indiana 46202; ^Department of Pediatrics, University of Chicago, Chicago, Illinois 60637; and ^^Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York USA

[Author Affiliation]

Received for publication 10 October 2000 and in final form 7 June 2001. Address reprint requests to Dr. Alonso P. Moreno, Krannert Institute of Cardiology, 1111 West 10th Street, Indianapolis, IN 46202. Tel.: 317-6306051; Fax: 317-630-7776; E-mail: amoreno@iupui.edu.