Abstract
Abstract: :
Purpose: The modulation of non–junctional currents by zinc has been demonstrated for connexons formed by perch Cx35, a vertebrate retinal connexin expressed in Xenopus oocytes, as well as for hemichannels formed by Cx38, a connexin endogenous to these oocytes (Chappell et al. 2003. Biol.Bull. 205:209–211). In this study we attempted to determine whether the ability of histidine to block the modulatory effects of zinc on hemichannel currents is due to chelation of ionic zinc (Zn2+), or a direct action of histidine on the hemichannel currents, or a combination of the two. Methods: The endogenous Cx38 hemichannels of Xenopus oocytes provide a useful model system with which to examine the modulatory effects of zinc and its regulation by histidine, a zinc chelator. Using the two–electrode voltage–clamp recording technique, we studied the effect zinc alone, or when superfused in the presence of histidine, on the currents mediated by the connexons formed by the endogenous connexin (Cx38) of defolliculated stage V–VI Xenopus oocytes. Results: Responses elicited by a series of 10 second pulses from a holding potential of –40 mV to +60 mV in 20 mV increments were obtained from oocytes superfused with a modified Barth’s (MB) solution containing 1 mM zinc, either alone or together with 1 mM histidine, and with histidine alone. Here we show that 1 mM zinc greatly suppressed the voltage–activated hemichannel currents, and that 1 mM histidine blocked completely the action of zinc. There was no evidence of a histidine–induced change in hemichannel currents when repeated in solutions to which 1 mM histidine had been added. When data were normalized to the +60 mV response obtained in the first control MB series, the pooled data from all control MB (n=10) and histidine (n=7) series obtained from four different oocytes were identical within +/– 1 SEM at all voltage steps from –20 to +60 mV. Conclusion: The block of the hemichannel response to ionic zinc by histidine is due solely to the chelation of zinc by histidine. Histidine alone has no effect on hemichannel currents mediated by the endogenous Cx38 connexons of frog oocytes. It appears likely that similar effects of histidine on zinc modulation of hemichannels expressed in the membranes of these oocytes or on the membranes of native retinal neurons may be interpreted as evidence for a role of ionic zinc, and not a direct effect of histidine on the channels themselves. We suggest that zinc chelation by histidine provides a viable method with which to study the effects of ionic zinc on retinal processes involving the activities of hemichannels and/or gap junctions formed by retinal connexins.
Keywords: gap junctions/coupling • pharmacology • retina