Structural bases for the chemical regulation of Connexin43 channelsRegulation of cardiac connexinsStructural bases for Cx43 regulation. The ball-and-chain hypothesisIdentification and characterization of connexin-interacting proteinsAntibody array: a screening method to search for potential molecular partnersKinetics, stoichiometry and amino acid identity in protein-protein interactionsEnzyme-Linked Sorbent Assay (ELSA)Surface plasmon resonanceMirror resonance spectroscopySedimentation equilibriumTranslational diffusion analysis15N-Heteronuclear Single Quantum Coherence (HSQC)Intra-molecular interactions in the chemical regulation of Cx43Structure of the Cx43 pore and pH gatingThe structure of the cytoplasmic loopThe carboxyl terminal domainInter-molecular interactionsConclusions and potential future directionsReferencesReviewStructural bases for the chemical regulation of Connexin43 channelsMario Delmara,*, Wanda Coombsa, Paul Sorgenb, Heather S. Duffyc, Steven M. TaffetdaDepartment of Pharmacology, SUNY Upstate Medical University, 766 Irving Ave, 13210 Syracuse, NY, USAbDepartment of Biochemistry and Molecular Biology, University of Nebraska Medical Center. Omaha, NE, USAcDepartment of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USAdDepartment of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY, USAReceived 7 October 2003; received in revised form 19 November 2003; accepted 10 December 2003Time for primary review 21 daysAbstractConnexins proteins associate with a variety of catalytic and non-catalytic molecules. Also, different domains of connexin can bind to eachother, providing a mechanism for channel regulation. Here, we review some of these associations, placing particular emphasis on theintramolecular interactions that regulate Connexin43 (Cx43). We also describe some novel methods that allow for the characterization ofprotein–protein interactions such as those observed in the cardiac gap junction protein Connexin43. Overall, intra- and inter-molecularinteractions may regulate gap junctions to filter the passage of molecular messages between cells at the appropriate time and between theappropriate cells. As a potential area for future investigations, we also speculate as to whether some of the inter-molecular interactionsinvolving connexins lead to modifications in the function of the associated protein, rather than on the function of connexin itself.D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.Keywords: Connexin; Gap junctions; Cx43; Gap junction regulationThe conv entional ‘‘textbook’’ picture of a gap junctionshows an o ligomeric complex of connexin proteins embed-ded in a lipid bilayer and making contact with a homologousstructure in the neighboring cell. This same picture wouldprobably show a rigid, hydrophilic pathway connecting bothcells and, if this is a cardiac electrophysiology text, it wouldnote that gap junctions provide a low-resistive pathway forthe transfer of electrical charges du ring action potentialpropagation. The latter picture is correct but incomplete.Recent studies suggest that connexins are not stand-aloneidle pores but rather highly dynamic structures that associatewith a variety of other catalyt ic [1] and non-cat alyticmolecules [2,3]. Gap junctions are required not only forthe electrical function of the heart [4] but also for biologicalprocesses, such as cardiac embryogenesis, that dependlargely on the synchronous function of non-excitable cells[5]. Here, we review some recent concepts on the molecularinteractions that take place within the microenvironment ofa gap junction. We place particular emphasis on the conceptthat interactions within the connexin molecules (i.e., intra-molecular interactions) may be essential for Cx43 regula-tion. These interactions may regulate the permeability andselectivity of the channel, as well as the ability of theconnexin molecules to interact with other partners. Giventhe growing interest in the study of protein–protein inter-actions as a mechanism of gap junction regulation, we alsodiscuss some novel methods that have been recently appliedto the identification and characterization of the inter- andintra-molecular interactions involving gap junct ion proteins.Molecular promiscuity may be a key mechanism by whichgap junctions filter the passage of information between cellsaccording to specific cues provided by changes in theintracellular space.1. Regulation of cardiac connexinsGiven the diversity of connexin isotypes, and theirheterology in terms of regulatory functions, we will focusthe present article on one particular isotype, Connexin43(Cx43). This is the most abundant connexin in the heart as0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.cardiores.2003.12.030* Corresponding author. Tel.: +1-315-464-7987; fax: +1-315-464-8014.E-mail address: [email protected] (M. Delmar).www.elsevier.com/locate/cardioresCardiovascular Research 62 (2004) 268–275well as other tissues such as brain. Moreover, there is a widevariety of regulatory mechanisms that can alte r the conduc-tivity of gap junctions [6]. A thorough review of all thosedifferent mechanisms is beyond the scope of this article.Here, we will review some of the molecular interactions thatoccur both inside a Cx43 molecule (intra-molecular) as wellas between connexins and other associated molecules (inter-molecular) and that can modify channel function.1.1. Struc tural bases for Cx43 regulation. The ‘‘ball-and-chain’’ hypothesisThere is general consensus that the carboxyl terminal(CT) regio n of Cx43 acts as the regulatory domain. Initialstudies showed that truncation of the CT domain at aminoacid 257 prevented acidification-induced uncoupling [7].That work was followed by a study indicating that uncou-pling could be restored if the CT domain was co-expressedas a separate fragment [8]. Based on these and other data,we proposed that pH regulation of Cx43 is consistent with a‘‘ball-and-chain’’ model of gating. This model, originallyproposed for voltage gating of the sodium channel [9] andlater demonstrated for the N-type inactivation of Shaker [10]and other potassium channels, states that the channel isblocked by the interaction between an intracellular particle(a ‘‘ball’’)—which is tethered t o a flexible element (a‘‘chain’’)—and a receptor