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1 Hunter College and The Graduate Center, CUNY, New York, NY
2 University of Illinois College of Medicine, Chicago, IL
* Corresponding author: harrripp@uic.edu
Connexins are a multigene family of structural proteins comprising gap-junctional channels, the aqueous pores that link electrically coupled cells in tissues throughout the body. These narrow passages (d 
16 Å) allow the intercellular exchange of ions, second messengers, and other small molecules having a molecular mass 
1 kDa. In the course of forming gap junctions, the connexins oligomerize into hexameric arrays known as "connexons" or "hemichannels" that assemble in the plasma membrane before docking with the connexons of adjacent cells (1). There is now abundant evidence that, at this penultimate stage of gap-junction formation, hemichannels can be activated both chemically and electrically (2,3), that their properties often reflect those of fully formed gap junctions (4), and that the modulation of hemichannel activity may be of physiological significance (5).
Light-induced changes in the chemical environment of the vertebrate retina have profound effects on both neurotransmitter-gated and electrical synapses; and these effects can, in turn, alter the sensitivity, receptive field organization, and signalling pathways that transmit the visual message to the CNS. One putative neuromodulator that has aroused a great deal of interest in recent years is zinc, which has been found in the synaptic vesicles of glutamatergic neurons in brain and retina (6). In the retinas of amphibia (7), fish (8), and mammals (6), zinc is present in photoreceptors, which signal second-order cells by regulating glutamate release in response to photic stimulation. Although the co-release of zinc with glutamate remains conjectural (9), the effects it exerts on retinal neurons have not been explored extensively (8,10); and no studies have addressed the question of its effect on connexins or the channels they form.
In the present study, we used the two-electrode voltage-clamp recording technique to examine the effects of zinc on the currents mediated by the connexons formed by the endogenous connexin (Cx38) of stage V-VI Xenopus oocytes, and those formed by perch Cx35, a connexin expressed in neurons of the vertebrate retina (11,12). To study the behavior of Cx35, cells were tested 48 to 72 h after they were injected with 46 nl of a mixture of 10 ng/cell Cx35 cRNA and 10 ng/cell of an antisense oligonucleotide to Cx38. The recordings were made with the cells bathed in a Na+-free medium to eliminate the large Na+-dependent currents that are similar in time course to the hemichannel currents, but of opposite polarity (13); zinc chloride was added without substitution. Responses were elicited with a series of 10-s pulses from a holding potential of -40 mV to +60 mV (see protocol, Fig. 1B inset), and were recorded with low resistance electrodes (0.7–1.5 M
) connected to a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA) and controlled by protocols generated in pClamp 8 (Axon). Data were analyzed in ClampFit (Axon) and plotted with software programs in Origin (Microcal Inc., Northampton, MA).
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+20 mV. After changing the bath solution to one containing 10 µM zinc, substantially greater hemichannel currents were elicited at these voltages (Fig. 1B). When the bathing medium was switched to one containing 1 mM zinc, the hemichannel currents were suppressed below those recorded in MB (Fig. 1C). The I-V data obtained with an oocyte expressing Cx35 for the range of zinc concentrations tested are illustrated in Figure 1D. It is evident that relatively low concentrations of zinc (1 µM and 10 µM) produce enhanced current responses at depolarizing voltages 40 mV, and the effect is reversed when the Zn concentration is increased to 100 µM. It should be noted that hemichannel currents recorded during experimental runs in which the solutions were delivered in reverse order (i.e., decreasing zinc concentrations from 1 mM zinc through 1 µM zinc to Na-free MB) gave rise to the same biphasic behavior. Comparable results were obtained with Cx38 (Fig. 1E), where we show, in addition, that the blocking effect of 1 mM zinc could be completely reversed by coapplication of 1 mM histidine, a zinc chelator. Hemichannel currents elicited by depolarizing voltage steps from -40 mV to +40 mV from all oocytes tested were normalized to their control current in Na-free MB solution and averaged according to connexin type for each concentration of zinc applied. The results from 5 Cx35 oocytes (Fig. 1F) and 11 Cx38 oocytes (Fig. 1G) were similar. Currents recorded in 1 and 10 µM zinc were greater than in the control (MB) solution, but decreased when the cells were bathed in 100 µM and 1 mM zinc. A return to MB (Fig. 1F) following a zinc series returned the hemichannel currents to their control values. Cells injected with the antisense to Cx38 alone (controls) did not exhibit significant hemichannel activity (results not shown). The biphasic effect of zinc on membrane currents is not unique. As shown in an earlier study (8), the addition of 10 µM zinc greatly enhanced GABA-induced currents mediated by GABAA receptors (GABAAR) of skate bipolar cells; in contrast, the currents were markedly reduced when the cells were exposed to 1 mM zinc. Moreover, the zinc enhancement of hemichannel currents appears to be insensitive to voltage; currents in 1 µM and 10 µM zinc recorded at +40 and +60 mV were approximately 1.7 times greater than in MB, a finding consistent with that obtained for GABAAR-mediated currents of bipolar cells. These observations suggest that zinc may interact with connexins at two external membrane binding sites, with very different affinities for zinc. The high affinity site, activated at low concentrations of zinc, gives rise to an enhancement of hemichannel currents, whereas the low affinity site requires high concentrations of zinc to produce its inhibitory effect.
The present findings, and evidence that zinc is located within the synaptic terminals of vertebrate photoreceptors (7,8), raise the possibility that zinc modulation of hemichannel activity contributes to the processing of visual information in the distal retina.
These studies were conducted at the Marine Biological Laboratory, Woods Hole, Massachusetts, and supported by grants from the National Eye Institute (EY-06516 and EY-01792), a PSC/CUNY grant (65711-0034) to RC, an unrestricted award to the UIC Department of Ophthalmology and Visual Sciences from Research to Prevent Blindness, Inc., a Senior Research Investigator Award from the RPB (HR), and an Award of Merit from the Alcon Research Institute (HR).
Literature Cited
This article has been cited by other articles:
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  R. L. Chappell, H. Qian, J. Zakevicius, and H. Ripps Histidine Suppresses Zinc Modulation of Connexin Hemichannels Biol. Bull., December 1, 2004; 207(3): 188 - 190. [Full Text] [PDF]
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S. Redenti and R. L. Chappell Zinc Chelation Enhances the Sensitivity of the ERG b-Wave in Dark-Adapted Skate Retina Biol. Bull., October 1, 2003; 205(2): 213 - 214. [Full Text] [PDF]
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