SUMMARY1. The kinetics of photoresponses to flashes and steps of light of rods, from the retina of the newt Triturus cristatus, were analysed by recording the membrane current with a suction electrode.2. In dark-adapted conditions the relation between the normalized amplitude of the photoresponse at a fixed time 1 s after the onset of light and the light intensity could be fitted by an exponential or a polynomial relation. In the presence of a steady bright light the same relation could be fitted by a Michaelis-Menten relation.3. The kinetics of photoresponses to light stimuli was reconstructed using a model in which: (i) three molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) open a light-sensitive channel; (ii) light activates the enzyme phosphodiesterase, which hydrolyses cyclic GMP, thus closing light-sensitive channels; (iii) Ca2" ions permeate through light-sensitive channels; and (iv) intracellular Ca21 inhibits, in a co-operative way, the enzyme cyclase, which synthesizes cyclic GMP. 4. The model reproduces the shortening of the time to peak of brief flash photoresponses from about 1080 ms to about 690 ms with brighter lights. The model also explains the shortening of the time to peak to 350 ms observed in the presence of a steady light and the lack of a further acceleration with brighter flashes of lights.5. The presence in the model of an intracellular calcium buffer accounts for the partial reactivation of the photocurrent following a step of light, lasting several seconds. The time course of this reactivation is not accelerated by a steady bright light both experimentally and in the model. 6. After the extinction to a long step of light the photocurrent showed a rapid partial reactivation, which was followed by a slow component of the photoresponse which extinguished with a rate constant of about 0 05 s-'. The model explains the origin of this slow component by assuming that the inactivation of excited rhodopsin is partially reversible. 7. The model is also able to explain the particular changes of kinetics when t To whom correspondence and reprint requests should be sent.

L-type and R-type Ca(2+) currents were detected in frog semicircular canal hair cells. The former was noninactivating and nifedipine-sensitive (5 microM); the latter, partially inactivated, was resistant to omega-conotoxin GVIA (5 microM), omega-conotoxin MVIIC (5 microM), and omega-agatoxin IVA (0.4 microM), but was sensitive to mibefradil (10 microM). Both currents were sensitive to Ni(2+) and Cd(2+) (>10 microM). In some cells the L-type current amplitude increased almost twofold upon repetitive stimulation, whereas the R-type current remained unaffected. Eventually, run-down occurred for both currents, but was prevented by the protease inhibitor calpastatin. The R-type current peak component ran down first, without changing its plateau, suggesting that two channel types generate the R-type current. This peak component appeared at -40 mV, reached a maximal value at -30 mV, and became undetectable for voltages > or =0 mV, suggestive of a novel transient current: its inactivation was indeed reversibly removed when Ba(2+) was the charge carrier. The L-type current and the R-type current plateau were appreciable at -60 mV and peaked at -20 mV: the former current did not reverse for voltages up to +60 mV, the latter reversed between +30 and +60 mV due to an outward Cs(+) current flowing through the same Ca(2+) channel. The physiological role of these currents on hair cell function is discussed.

SUMMARY1. Properties of a new preparation for studying the physiology and biochemistry of phototransduction in retinal rods are described. Whole-cell voltage clamp was used to record the generation, maintenance and light-sensitivity of dark current in rod outer segments that had been isolated from the rest of the receptor cell by detachment at the connecting cilium.2. Detached outer segments dialysed with standard internal solution supplemented with physiological amounts of ATP (5 mM) and GTP (1 mM) developed a standing inward dark current that was the sum of three components: -91 % light-sensitive current, -6 % Na'-Ca2+, K+ exchange current and 3 % leakage current. Light-sensitive dark current (mean amplitude --63 pA) was suppressed transiently by brief flashes in an intensity-dependent manner. Light responses had the same kinetics, sensitivity and intensity-response relationship as those recorded from intact rods.3. Dialysed outer segments differed from intact rods in that intense flashes evoked saturating responses that recovered incompletely to a plateau of reduced dark current caused by incomplete inactivation of the transduction cascade. Light sensitivity was reduced for a short time following an intense flash and then recovered despite persistent reduction of dark current. This suggests that there is no fixed relationship between dark current amplitude and light sensitivity.4. Light-sensitive dark current faded rapidly when outer segments were not supplied with nucleotides. Outer segments dialysed with solution that contained cyclic GMP, but no ATP or GTP, supported dark current at a level that increased with [cyclic GMP]. When basal phosphodiesterase (PDE) activity is inhibited, 8 /SM cyclic GMP supports a dark current of -70 pA.5. Light sensitivity decreased during recordings made with solution that contained only cyclic GMP, consistent with the inhibition of G protein activation by loss of GTP. After thorough nucleoside triphosphate depletion, however, intense

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na(+):Ca(2+),K(+) exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K(+), Na(+) or Ca(2+)), the OS membrane resistance (R ( m )) was typically larger than 1 GOmega in the presence of 1 mM external Ca(2+). Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in approximately 50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.

Crystal clear: A detailed conformational characterization of a synthetic analogue of the peptide antibiotic alamethicin (see structure; C gray, N blue, O red) has been achieved by X‐ray diffraction. The high‐resolution structure of this analogue, which incorporates a spin probe, paves the way for a better understanding of the mode of action of alamethicin.