DIFFERENCES IN CALCIUM HOMEOSTASIS BETWEEN RETINAL ROD AND CONE PHOTORECEPTORS REVEALED BY THE EFFECTS OF VOLTAGE ON THE CGMP-GATED CONDUCTANCE IN INTACT-CELLS

We measured currents under voltage clamp in intact retinal rod photore ceptors with tight seal electrodes in the perforated patch mode. In th e dark, membrane depolarization to voltages greater than or equal to 20 mV activates a time- and voltage-dependent outward current in the o uter segment. This dark voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course that is voltage dependent. DVAC reaches its maximum enhancement of similar to 30% in 4-6 s at +60 mV. DVAC is entirely suppressed by light and its current-voltage curve an d reversal potential are the same as those of the photocurrent. Theref ore, DVAC arises from the opening in darkness of the cGMP-gated channe ls of the outer segment. DVAC is blocked by BAPTA loaded into the cell 's cytoplasm and is enhanced by lowering extracellular Ca2+ concentrat ion. Because the cCMP-gated channels are not directly gated by voltage and because BAPTA blocks DVAC, we suggest this signal arises from a v oltage-dependent decrease in cytoplasmic Ca2+ concentration that, in t urn, activates guanylyl cyclase and causes cGMP synthesis. In rods loa ded with high cytoplasmic Na+, membrane depolarization in darkness to voltages greater than or equal to +20 mV inactivates the outward curre nt in the outer segment with an exponential time course. We call this DVIC (dark, voltage-inactivated current). DVIC reflects voltage-depend ent closing of the cGMP-gated channel in the dark. DVIC, too, is block ed by cytoplasmic BAPTA, and it arises from a voltage-dependent rise i n cytoplasmic Ca2+ in darkness, which occurs only if cytoplasmic Na is high. We develop a quantitative model to calculate the rate and exten t of the voltage-dependent change in cytoplasmic Ca2+ concentration in a normal rod. We assume that this concentration is controlled by the balance between Ca2+ influx through the cGMP-gated channels and its ef flux through a Na+/Ca2+, K+ exchanger. Lowered cytoplasmic Ca2+ is lin ked to guanylyl cyclase activation with characteristics determined fro m biochemical studies. The model considers the cytoplasmic buffering o f both Ca2+ and cGMP. Simulated data generated by the model fit well D VAC measured in rods and also DVAC previously measured in cones. DVAC in cones is larger in magnitude and faster in time course than that in rods. The successful fit of DVAC by the model leads us to suggest tha t the activity and Ca2+ dependence of the enzymes of transduction are not different in rods and cones, but the quantitative features of Ca2 homeostasis in the outer segment of the two receptor types differ pro foundly. In general, for a given change in outer segment current, whet her caused by light or by voltage, the changes in cytoplasmic Ca2+ are larger and faster in cones than in rods. This difference reflects spe cific differences between receptor types in their outer segment volume as well as in the relative fraction of the current carried by Ca2+ th rough the cGMP-gated channels, the intracellular Ca2+ buffering capaci ty and the rate of Ca2+ clearance from the outer segment by the Na+/Ca 2+, K+ exchanger.