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. Voltageclamp
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 connexinspecific
effects on multichannel and single-channel properties.
The Cx43-tag was most affected and the Cx45-tag,
least. The modifications included (1) Vj-sensitive gating of
Ij (Vj, gap junction voltage; Ij, gap junction current), (2)
contribution and (3) kinetics of Ij deactivation and (4)
single-channel conductance. The first three reflect alterations
of fast Vj 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.