Dark Ionic Flux and the Effects of Light in Isolated Rod Outer Segments

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Abstract
We have determined the permeability properties of freshly isolated frog rod outer segments by observing their osmotic behavior in a simple continuous flow apparatus. Outer segments obtained by gently shaking a retina are sensitive but nonideal osmometers; a small restoring force prevents them from shrinking or swelling quite as much as expected for ideal behavior. We find that Na+, Cl-, No3-, glycerol, acetate, and ammonium rapidly enter the outer segment, but K+, SO4=, and melezitose appear impermeable. The Na flux is rectified; for concentration gradients in the physiological range, 2 x 109 Na+ ions/sec enter the outer segment, but we detect no efflux of Na+, under our conditions, when the gradient is reversed. Illumination of the outer segment produces a specific increase in the resistance to Na+ influx, but has no effect on the flux of other solutes. This light-dependent Na+ resistance increases linearly with the number of rhodopsin molecules bleached. We find that excitation of a single rhodopsin molecule produces a transient (∼1 sec) "photoresistance" which reduces the Na+ influx by about 1%, thus preventing the entry of about 107 Na+ ions. At considerably higher light levels, a stable afterimage resistance appears which reduces the Na influx by one-half when 106 rhodopsin molecules are bleached per rod. We have incorporated these findings into a model for the electrophysiological characteristics of the receptor.