The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.
According to the classical calcium hypothesis of synaptic transmission, the release of neurotransmitter from presynaptic terminals occurs through an exocytotic process triggered by depolarization-induced presynaptic calcium influx. However, evidence has been accumulating in the last two decades indicating that, in many preparations, synaptic transmitter release can persist or even increase when calcium is omitted from the perfusing saline, leading to the notion of a "calcium-independent release" mechanism. Co2+, Ni2+, and Zn2+ is to a large extent accounted for by a depolarizing shift of the Ca2+ current activation curve, by means of an interaction with the negative fixed charges on the photoreceptor presynaptic membrane. Presynaptic depolarization with extrinsic currents, or reduction of extracellular Ca2+, relieves the transmission block because these conditions bring presynaptic transmembrane potential to levels where Ca>2 current can still be activated. Our results may give new insights into the problem of so-called "calcium-independent synaptic transmission" (6, 7). MATERIALS AND METHODSLight Responses in Retinal Eyecups. Eyecups were prepared from the retina of turtles (Pseudemys scripta elegans) or salamanders (Ambystoma tigrinum) and continuously superfused with a modified Ringer saline bubbled continuously with a mixture of 95% 02 and 5% Co2. The composition (in mM) of the saline used in turtle experiments was the following: NaCl, 110; KCI, 2.6; NaHCO3, 22; MgCl2, 2; CaCl2, 2; Dglucose, 10 (pH 7.4). Salamander saline had the followingThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.composition: NaCl, 95; KCI, 2.5; NaHCO3, 30; MgCl2, 2; CaCl2, 2; D-glucose, 10 (pH 7.6). Intracellular recording of the light responses induced by 5-mm-diameter white light spots of variable intensity and duration were made in HCs (and in other retinal neurons) by using fine tip microelectrodes prepared with a Brown-Flaming puller (Sutter Instruments, Novato, CA). The concentration of Ca2> in the nominally zero Ca2> Ringer solution, without EGTA added, was less than 10 ,uM. The pH of the solutions containing EGTA or EDTA was readjusted with NaOH. The concentration of Ca2> in media containing CaCl2 in variable proportions and EGTA (2-10 ,uM) or EDTA (2-5 ,.tM) ranged from 0.45 to 2.4 nM and was estimated by using the computer program SOL i.d. written by Eric Ertel (Hoffmann-La Roche). To keep a normal concentration of Mg2+ in media containing EGTA the proportion of MgCl2 in the perfusing saline was adjusted accordingly. Chemicals were obtained from Sigma. Bicuculline was used as the soluble methobromide salt and added to the perfusing medium just before application to the retina. Picrotoxin was first dissolved in slightly basic, hot distilled water and then added to the Ringer solution. Neutral density filters were used to attenuate light intensity. The flux de...
An M-like K+ current (IKx) helps set the rod photoreceptor resting potential and accelerates the response to dim light. Recorded with ruptured-patch whole cell techniques, the amplitude of IKx diminished, and activation occurred at increasingly negative potentials as a function of time. In contrast, IKx was stable during nystatin perforated-patch recording. Stability during ruptured-patch recording could be induced by raising the intracellular Ca2+ concentration ([Ca2+]i) or by including caffeine or D-myo-inositol 1,4,5-trisphosphate in the pipette. This Ca(2+)-induced stability of IKx was blocked by inhibitors of Ca2+/calmodulin-dependent protein kinases, such as W-7, KN-62, chelerythrine, or H-7. Okadaic acid, an inhibitor of protein phosphatases, maintained IKx stability even at low [Ca2+]i. The requirement for phosphorylation was demonstrated by depleting MgATP or by providing 5'-adenylylimidophosphate, a nonhydrolyzable analog of ATP, either of which blocked the Ca(2+)-induced stability of IKx. These observations show that phosphorylation regulates IKx and that a stimulus controlling this action is [Ca2+]i. Should [Ca2+]i change during light adaptation, changes in IKx might alter the resting potential and temporal response properties of rod photoreceptors.
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