HEMI-GAP-JUNCTION CHANNELS IN SOLITARY HORIZONTAL CELLS OF THE CATFISH RETINA

1. Solitary horizontal cells were isolated from catfish retinas and their membrane current was recorded with a whole-cell voltage clamp. Reducing the extracellular Ca2+ concentration produced a current that could be suppressed by dopamine. This Ca2+- and dopamine-sensitive current is hereafter termed I(gamma). The voltage dependence, cytoplasmic regulation, and permeability of the I(gamma) channel suggest that it is half of a gap-junction channel. 2. I(gamma) was voltage and time dependent. In the steady state, the current-voltage relation displayed outward rectification at voltages more depolarized than 0 mV and a negative resistance region at voltages more hyperpolarized than -15 mV. The reversal potential was 3.3 +/- 1.5 mV when NaCl was the predominant extracellular salt and potassium-D-aspartate was the predominant intracellular salt. 3. The size of I(gamma) depended on the extracellular Ca2+ concentration. I(gamma) was maximal at external Ca2+ concentrations below 10-mu-M, half-maximal at 220-mu-M-Ca2+, and reduced to less than 4% of its maximum amplitude at external Ca2+ concentrations above 1 mM. Increasing the extracellular Ca2+ concentration reduced the amplitude of I(gamma) without changing the shape of the current-voltage relation or the kinetics of inactivation. Thus, rectification does not result from a voltage-dependent block by extracellular Ca2+. 4. Patches of cell membrane were voltage clamped in both the cell-attached and excised-patch configurations. In the cell-attached configuration, the addition of dopamine to the solution outside the patch pipette blocked the opening of channels within the membrane patch. Thus, dopamine closes I(gamma) channels by initiating an intracellular messenger cascade. In the excised-patch configuration, a maximum conductance of 145 pS was measured while Cs+ and tetraethylammonium+ (TEA+) were the only monovalent cations on both sides of the membrane. 5. The ability of dopamine to suppress I(gamma) was blocked by introducing an inhibitor of the cyclic AMP-dependent protein kinase, PKI5-24, into the cytoplasm. Thus, the action of dopamine is mediated by a pathway that includes the activation of a cyclic AMP-dependent kinase. 6. I(gamma) was suppressed by nitroprusside, an agent which activates guanylate cyclase and increases the intracellular cyclic GMP concentration. The effect of nitroprusside was not altered by the intracellular application of PKI5-24. Thus, nitroprusside suppresses I(gamma) through a pathway that does not include the activation of a cyclic AMP-dependent kinase. 7. Intracellular acidification also suppressed I(gamma). 8. The gap junction between adjacent horizontal cells is modulated by cyclic AMP, cyclic GMP, and intracellular pH. I(gamma) and junctional conductance were measured simultaneously in cell pairs. The three intracellular messengers produced co-ordinated changes in I(gamma) and junctional conductance. 9. Junctional current was measured while transjunctional voltage was stepped to values between +/- 115 mV. The voltage- and time-dependent behaviour of the gap junction could be predicted from the observed voltage- and time-dependent behaviour of I(gamma) by making two assumptions: first, I(gamma) is produced by hemi-gap-junction channels; and second, the complete gap junction is formed by the end-to-end apposition of two hemi-gap-junction channels, each responding independently to half of the transjunctional voltage field. 10. Lucifer Yellow CH entered solitary horizontal cells bathed in a medium containing a reduced Ca2+ concentration. Adding dopamine to the extracellular solution or increasing the extracellular Ca2+ concentration blocked the influx of dye. Thus, large solutes, known to permeate gap junctions, also permeate through the channel that conducts I(gamma). 11. We conclude that I(gamma) flows through hemi-gap-junction channels expressed in the surface membrane. Furthermore, the end-to-end apposition of two hemi-gap-junction channels produces a gap junction in which each hemi-channel responds independently to transjunctional voltage.