Format

Send to

Choose Destination
J Membr Biol. 2011 Apr;240(3):139-50. doi: 10.1007/s00232-011-9352-z. Epub 2011 Mar 19.

Influence of v5/6-His tag on the properties of gap junction channels composed of connexin43, connexin40 or connexin45.

Author information

1
Institute of Physiology, University of Bern, Bühlplatz 5, 3012 Bern, Switzerland.

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.

PMID:
21424225
PMCID:
PMC3069306
DOI:
10.1007/s00232-011-9352-z
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Springer Icon for PubMed Central
Loading ...
Support Center