Outer segments of rod photoreceptors with the attached ellipsoid region of the inner segment were isolated from Rana pipiens retinae, and their membrane photocurrents measured with the suction electrode technique in the 'ellipsoid-in' configuration. Under dark adapted conditions in standard Ringer solution, isolated rod outer segments with ellipsoids exhibited maximal photocurrents of 10-30 pA, and light sensitivities of 0.2-1.0 pA/isomerization. A local perfusion technique was employed to change rapidly the solution bathing the outer segment. Rods were tested for their sensitivity to protons by perfusion with Ringer solution of altered pH. The dark current was reversibly suppressed by low pH: in Ringer solution with Calcium activity a(Ca) = 10-3 M dark current suppression obeyed a hyperbolic saturation law with apparent dissociation constant, pK(a) = 4.8. The decay of dark current of rods following poisoning with ouabain was retarded by low pH perfusion, as it was by light. Protons thus act to suppress the outer segment Na+ conductance. Three experiments support the hypothesis that protons act interior to the plasma membrane in suppressing the dark current. (1) Perfusion of rods at constant pH with Ringer solution having increased CO2 suppressed the dark current. (2) Removal of perfusate containing 50 mM-NH4Cl causes transient dark current suppression. (3) Acetate, which acts as a neutral proton carrier, when added to Ringer solution, shifts the apparent pK(a) of dark current suppression to a higher pH. Dark current suppression by protons and recovery occurred with a time constant of ca. 1 s. Low pH perfusion retarded the recovery of the dark current from a saturating flash, slowed the light response in its linear range, and increased light sensitivity. Perfusion at pH = 10.5 caused a slight increase in dark current, sped up the recovery of the rod from a saturating flash, accelerated the linear response and decreased the light sensitivity. Lowering a(Ca) of the Ringer solution caused the proton sensitivity of the dark current to drop. At a(Ca) = 5 x 10-6 M the apparent pK(a) of dark current suppression was shifted about 0.8 pH units to pH = 4.0. Cells at a(Ca) = 10-9 M were insensitive to pH = 3.5, which completely suppressed the dark current at normal a(Ca). Lowered a(Ca) decreased light sensitivity. Both proton sensitivity and light sensitivity of dark current suppression were estimated for each member of a population of rods in various a(Ca)s: proton sensitivity and light sensitivity were found to be linearly correlated over a range of 3 log units. It is estimated that an acid perfusion that would suppress 3% of the dark current (the average fraction suppressed by single isomerization) in 1 s would introduce about 105 protons into the outer segment. Calculations based upon in vitro data suggest that single isomerization can lead, by light-activated phosphodiesterase-catalysed hydrolysis of cyclic GMP, to production of more than 3 x 104 protons in 1 s. It is therefore proposed that protons produced by cyclic GMP hydrolysis constitute, via H+/Ca2+ exchange, a link in excitation.
|Original language||English (US)|
|Number of pages||26|
|Journal||Journal of Physiology|
|State||Published - 1984|
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