Control Of Sensitivity Following Pigment Bleaching By NADPH In Salamander Rods

Abstract

The recovery of sensitivity following photopigment bleaching requires the quenching of phototransduction, and the reduction of all-trans retinal is key. Retinol fluorescence increases after bleaching as a base to tip gradient in the rod outer segment and broadly matches the recovery of sensitivity. This gradient must result from a key component in retinal reduction, and we sought to determine how NADPH limits this process. Rod outer segment currents were recorded with suction electrodes, and responses were evoked by brief full-field flashes or by a narrow slit to stimulate selectively the base or tip of the outer segment. Simultaneous whole-cell recordings were made prior to bleaching to dialyze the cell with NADPH and track the recovery of sensitivity. After a 50% bleach rods remained in saturation for ∼ 12 minutes. The base recovered sensitivity with tau ∼ 160 s, but the tip recovered with tau ∼ 450 s resulting in a tip-base tau ratio of ∼ 3. Dialysis of 5 mM NADPH accelerated the recovery time by ∼ 2 min, and eliminated the tip-base difference. Dialysis with 1.66 mM NADPH didn't influence recovery and failed to eliminate the tip-base difference. After a 90% bleach rods remained saturated for ∼ 20 minutes with tip-base ratio ∼ 10 (base tau ∼ 250 s and tip tau ∼ 2700 s). Thus 5 mM NADPH eliminates the gradient along the outer segment, while 1.66 mM fails to influence the recovery of sensitivity; suggesting intrinsic NADPH exceeds 1.66 mM. In addition, tau at the base following 50% or 90% bleach are remarkably equivalent, suggesting that NADPH availability is sufficient to reduce all-trans retinal. The slower tip tau following 90% bleach suggests NADPH originates predominantly near the base, which is adjacent to mitochondria.