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Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions

Abstract

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.

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Figure 1: Schematic diagrams of a standard connexin molecule and gap junction channel.
Figure 2: Voltage sensitivity of macroscopic junctional currents in Cx36 and Cx50 transfected into N2A cells.
Figure 3: Single channel conductances of gap junction channels formed by Cx50 measured with step voltage pulse protocols.
Figure 4: The patch clamp workstation.
Figure 5: A fibroblast cell line transiently transfected with vector encoding Cx43-GFP using Lipofectamine 2000 and observed 24 h after transfection.
Figure 6: Simulation of steps involved in achieving the whole cell configuration of patch clamp recording.
Figure 7: DIC image of a pair of N2A cells imaged during dual whole cell patch clamp experiment.
Figure 8: Single-channel properties of heterotypic Cx46-Cx50 channels assessed by a ramp protocol.
Figure 9: Flufenamic acid (FFA) reduces junctional conductance in a reversible, concentration-dependent manner.

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Acknowledgements

We gratefully acknowledge the many talented graduate students and post doctoral fellows who have worked with us over the years since voltage clamping was first applied to gap junctions. Our research has been supported by grants from the National Institutes of Health (NIH) to D.C.S. and colleagues as well as foreign grants to investigators visiting the laboratory from other countries.

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Correspondence to David C Spray.

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del Corsso, C., Srinivas, M., Urban-Maldonado, M. et al. Transfection of mammalian cells with connexins and measurement of voltage sensitivity of their gap junctions. Nat Protoc 1, 1799–1809 (2006). https://doi.org/10.1038/nprot.2006.266

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