title: Influence of v5/6-His tag on the properties of gap junction channels composed of connexin43, connexin40 or connexin45.
abstract: HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V(j)-sensitive gating of I(j) (V(j), gap junction voltage; I(j), gap junction current), (2) contribution and (3) kinetics of I(j) deactivation and (4) single-channel conductance. The first three reflect alterations of fast V(j) gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.
authors.affiliation: Institute of Physiology, University of Bern, Bühlplatz 5, 3012 Bern, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Desplantez
authors.forename: Deborah
authors.initials: D
authors.lastname: Halliday
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Dupont
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Severs
authors.forename: Robert
authors.initials: R
authors.lastname: Weingart
mesh_terms: D015153:Blotting, Western; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000097003:Gap Junction alpha-5 Protein
chemical_list: D018031:Connexin 43; D017630:Connexins; D011994:Recombinant Proteins; D000097003:Gap Junction alpha-5 Protein; C082920:connexin 45