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Review
. 2014 Apr 17;588(8):1372-8.
doi: 10.1016/j.febslet.2013.12.023. Epub 2014 Jan 14.

Connexin hemichannel and pannexin channel electrophysiology: how do they differ?

Dakshesh Patel  1 , Xian Zhang  1 , Richard D Veenstra  2
Affiliations
Review

Connexin hemichannel and pannexin channel electrophysiology: how do they differ?

Dakshesh Patel et al. FEBS Lett. .

Abstract

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca(2+)]o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X7 receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X7 expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.

Keywords: Channels; Connexin; Gap junction; Hemichannel; P2X(7) receptor; Pannexin.

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Figures

Figure 1
Figure 1
A, An immunoblot for Cx43 showing a variable amount of endogenous Cx43 expression in HEK293 cells obtained from two different research laboratories. B, An immunoblot for Cx43 from the low Cx43 expressing HEK293-2 cells, a stable clone (80-1) of the same cell line after knockdown of Cx43 with an anti-Cx43 shRNA, and a scrambled control shRNA. Tubulin was used as a loading control. C, Quantification of the relative Cx43 expression levels from the HEK-2 cells, the Cx43 shRNA HEK-2 cell clone, and the scrambled shRNA control from three immunoblots like the one displayed in panel B. D, Anti-Cx43 antibody (Millipore #AB1728) was immunolocalized in HEK-2 cells with an Alexa Fluor555 conjugated secondary antibody. Gap junctions appeared as small puncta (arrows) located between adjacent HEK293 cells.
Figure 2
Figure 2
A, A whole cell patch clamp recordings from an HEK293-2 cell during repeated runs of a −40 to +30 mV voltage step under low (nominally zero) [Ca2+]o conditions. Activation of a membrane channel that remained open upon return to −40 mV was frequently evident. B, A whole cell recording from the same HEK-2 cell after the addition of 2 mM CaCl2 to the bath saline illustrates the reduction in observed endogenous channel activity. C, Ensemble averages of the five current traces displayed in panels A and B illustrating the difference in whole cell currents between 0 and 2 mM [Ca2+]o. D, The junctional current – voltage relationship acquired from this HEK-2 cell coupled to a partner HEK-2 cell via Cx43 gap junctions. The ratio of [Ca2+]o-sensitive nonjunctional membrane current to gap junction current was 0.04 in this HEK-2 cell pair.
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