RESUMO
1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate. 2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system. 3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels. 4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina.
Assuntos
Conexinas/fisiologia , Oócitos/fisiologia , Animais , Canais de Cloreto/fisiologia , Conexina 26 , Conexinas/efeitos dos fármacos , Conexinas/genética , Feminino , Canais Iônicos/genética , Potenciais da Membrana , Quinina/farmacologia , Xenopus laevisRESUMO
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.