Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions.
Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels.
del Corsso
Cristiane
C
The Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, 1410 Pelham Parkway South, Bronx, New York 10461, USA.
Srinivas
Miduturu
M
Urban-Maldonado
Marcia
M
Moreno
Alonso P
AP
Fort
Alfredo G
AG
Fishman
Glenn I
GI
Spray
David C
DC
D000818:Animals; D045744:Cell Line, Tumor; D017630:Connexins / Q000235:genetics / Q000502:physiology*; D017629:Gap Junctions / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D018408:Patch-Clamp Techniques; D051381:Rats; D014162:Transfection / Q000379:methods*; D014982:Xenopus laevis
D017630:Connexins