J Neurosci Methods 2002 Nov 15;1211:81-92. doi: 10.1016/s0165-0270(02)00243-1.

Pharmacological enhancement of hemi-gap-junctional currents in Xenopus oocytes.

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Hemichannels formed by expressing connexin subunits in Xenopus oocytes provide a valuable tool for revealing the gating properties of intercellular gap junctions in electrically coupled cells. We used the two electrode voltage-clamp technique to demonstrate that activation of the time-dependent outward hemichannel currents brings into play a sodium current of similar time course and opposite polarity; the interaction between these opposing currents had not been explored previously. Using the endogenous connexin (Cx38) of Xenopus oocytes as a model system, we have shown that substituting choline for sodium in the bath solution eliminates the sodium current, thereby unmasking large hemichannel currents, and enabling pharmacological studies of agents that are known to modulate gap-junctional conductances. The cinchona alkaloid quinine also effectively blocked the inward current, and in addition, enhanced significantly the Cx38 hemichannel currents in a dose-dependent fashion; the Hill coefficient of 1.9 suggests that the binding of at least two molecules of quinine is required to produce the effect. Intracellular quinine had no effect on hemichannel currents, and experiments on the displacement of quinine suggest that binding is at an external site near or within the mouth of the hemichannel. Intracellular acidification suppressed the quinine-enhanced hemichannel currents, indicating that quinine does not block the proton binding site. We found that retinoic acid (RA) and carbenoxolone, agents that block gap-junctional channels in coupled neurons and other cell types, also suppressed Cx38 hemichannel currents with an IC(50) of approximately 2 and 34 microM for RA and carbenoxolone, respectively. Raising extracellular calcium to 3 mM suppressed both the hemichannel current and the inward sodium current. These results provide a foundation upon which to further characterize the gating of hemichannel currents mediated by connexins expressed in Xenopus oocytes.

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Species referenced: Xenopus
Genes referenced: gja4.2