Single photon responses in Drosophila photoreceptors and their regulation by Ca2+.

摘要

1. Discrete events (quantum bumps) elicited by dim light were analysed in whole-cell voltage clamp of photoreceptors from dissociated Drosophila ommatidia. Bumps were automatically detected and analysed for amplitude, rise and decay times, and latency. 2. The bump interval and amplitude distributions, and the 'frequency of seeing' curve conformed to Poisson predictions for the absorption of single photons. 3. At resting potential (-70 mV), bumps averaged 10 pA in peak amplitude with a half-width of ca 20 ms, representing simultaneous activation of ca 15 channels. 4. The macroscopic response to flashes containing up to at least 750 photons were predicted by the linear summation of quantum bumps convolved with their latency dispersion. 5. Bump duration was unaffected by lowering the extracellular Ca2+ concentration ([Ca2+]o) from 1.5 to 0.5 mM, but increased >10-fold between 0.5 mM Ca2+ and 0 Ca2+. Bump amplitude was constant over the range 1.5-100 microM, but decreased ca 5- to 10-fold at lower Ca2+ concentrations. Bump latency increased by ca 50 % between 1.5 mM and 100 microM Ca2+o but returned to near control levels in Ca2+-free solutions. At intermediate [Ca2+]o bumps were biphasic with a slow rising phase followed by rapid amplification and inactivation. This behaviour was mimicked in high [Ca2+]o by internal buffering with BAPTA, but not EGTA. This suggests that Ca2+ influx through the light-sensitive channels must first raise cytosolic Ca2+ to a threshold level before initiating a cycle of positive and negative feedback mediated by molecular targets within the same microvillus. Quantum bumps in trp mutants lacking the major class of light-sensitive channel were reduced in size (mean 3.5 pA) representing simultaneous activation of only one or two channels; however, a second rarer (10 %) class of large bump had an amplitude similar to wild-type (WT) bumps. Bumps in trpl mutants lacking the second class of light-sensitive channel were very similar to WT bumps, but with slightly slower decay times. In InaDP215 mutants, in which the association of the TRP channels with the INAD scaffolding molecule is disrupted, bumps showed a defect in quantum bump termination, but their amplitudes and latencies were near normal.