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

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.