Effects of injections of calcium and EGTA into the outer segments of retinal rods of Bufo marinus.

1. Intracellular recordings were made from the outer segments of rods in the isolated, superfused retina of Bufo marinus. Cells were impaled under observation with a compound microscope fitted with an infra-red image converter. Changes of membrane voltage and some concomitant changes of input resistance were measured in response to light, membrane polarization and iontophoretic injections.2. By means of a double barrel micropipette, charge was passed into a rod from a micropipette barrel that contained Ca(2+) while no net current crossed the plasma membrane. In about half the cells, immediately after the injection, a hyperpolarization was observed that decayed with a time course similar to the decay of the receptor potential.3. Membrane hyperpolarization also occurred after a depolarizing current stopped flowing into a rod through a single barrel pipette that contained only K-acetate. This hyperpolarization was accompanied by an increase of membrane conductance. The reversal potential for the conductance-increase was between the voltage in the dark and the voltage in the absence of [Na(+)](out). A larger hyperpolarization became evident after an equal depolarizing current stopped flowing into a rod through a pipette that also contained Ca(2+); this larger after-hyperpolarization was due to both the cessation of depolarizing current and the injection of Ca(2+).4. A depolarization of 10-20 mV that lasted 2-60 sec became evident after hyperpolarizing current stopped flowing into a rod through a single-barrel pipette filled with K-EGTA. During the after-depolarization, the responses to small, dim spots of light were attenuated. No depolarization was observed after passing hyperpolarizing currents into rods through pipettes that contained either acetate(-), SO(2-) (4) or MOPS(-).5. These results show that sequestration of [Ca(2+)](in) depolarizes the plasma membrane and that an increase in [Ca(2+)](in) hyperpolarizes the membrane mimicking the later part of the receptor potential. These findings support the hypothesis of Yoshikami & Hagins (1971) that Ca(2+) is an intracellular messenger for excitation in vertebrate rods.