Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45 |
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Authors: | Thomas Desplantez Deborah Halliday Emmanuel Dupont Nicholas J Severs Robert Weingart |
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Institution: | 1.Institute of Physiology,University of Bern,Bern,Switzerland;2.National Heart and Lung Institute,Imperial College London,London,UK;3.Post Graduate School of Medicine, Faculty of Health and Medical Sciences,University of Surrey Guildford,Surrey,UK |
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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. |
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