Inhibition of phospholipase C by U-73122 blocks one component of the receptor current in Limulus photoreceptor

In the ventral nerve photoreceptor of Limulus a short, intense flash evokes a receptor current consisting of three components. In contrast to other hypotheses, we suggested previously that only the second component of the current is activated by the phospholipase C pathway, which releases calcium from intracellular stores by inositol trisphosphate. The present paper gives further evidence to our suggestion. It is demonstrated that U-73122, a specific inhibitor for the phospholipase C, selectively blocks the second component. The first and third components are moderately affected and could still be activated after the complete block of the second one. Results support the idea that the first and third components areactivated by pathways operating independently of phospholipase C.

Disturbing Gtp-Binding Protein Function Through Microinjection Into The Visual Cell Of Limulus

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4 A) against the Gɑ subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with Gɑ in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADPribosyltransferase C 3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GD P-β-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations

Single Photon-Evoked Events Of The Ventral Nerve Photoreceptor Cell Of Limulus Facilitation, Adaptation And Dependence Of Lowered External Calcium

Bumps, the elementary excitatory events of the Limulus ventral nerve photo receptor following a weak flash of light were recorded under voltage clamp conditions. The statistical distribution of various bump parameters and their changes caused by weak conditioning pre-illumination are described, and the influence of lowered external Ca2+-concentration together with normal or raised Mg2+-concentration (15 °C).1) Weak conditioning pre-illumination causes desensitization: the bump current amplitude, bump duration , bump area (current-integral), and the bump latency are diminished, the more, the stronger the conditioning flash, i.e. the light adaptation. Very weak conditioning pre-illumination causes facilitation, expressed by an increase in number and size of the observed bumps. The average bump latency, however, is already shortened under these conditions.2) Lowering the external Ca2+-concentration from 10 mmol/l to 250 (µmol/1 has its primary effect on the dark -adapted photoreceptor (without substantially reducing the ability for light adaptation ). It causes the following average changes: the amplitudes, durations, current-integrals, and the latencies of current bumps are greatly enlarged and the number of bumps is raised.3) Raised magnesium concentration from 50 to 100 mmol/l can partially compensate for the lack of calcium ; however, it enhances the effect of calcium deficiency on the latency, i.e. it further enlarges the average latencies. The results can be explained on the basis of our model of bump generation by two assumptions.1) Lowering the external calcium concentration causes a decrease in the cytosolic Ca2+-level without substantially reducing the intracellular calcium stores from which the light-adapting calcium release is fed. The lowered cytosolic Ca2+-concentration induces an “extra” dark adaptation resulting in greater bumps and more bumps exceeding the threshold of recognition. The bump latency, however, which behaves differently from all other bump parameters, is determined by a separate calcium -dependent reaction where magnesium competes with calcium antagonistically. 2) Facilitation is due to cooperativity of transmitter binding in order to open the ion channels

Stochastic Treatment of Bump Latency and Temporal Overlapping in Limulus Ventral Photoreceptors

We present quantum bumps obtained from flash experiments at the Limulus ventral nerve photoreceptor under voltage clamp conditions. The results are shown and discussed in form of histograms for the latency, amplitude and net charge transfer (current time integral) of the bump current responses. We argue that the experimental latency histogram s cannot be described satisfactorily by chemical models if one assumes that not more than one photon is captured per flash. Instead of, one has to take into account the Poisson statistics of the captures of 0,1,2 ,... photons released by a single flash. We show that the inclusion of Poisson statistics makes the effective latency histograms of flash responses typically asymmetric and skewed to wards short latencies as compared to that of model histograms for one-photon responses. Our conjecture also implies that under our experimental conditions a fraction of up to 20% of the bump responses evoked by a flash should be suspected to be superpositions of two ore more one-photon responses which cannot be separated by any kind of evaluation analysis. Consequently, the average values of amplitudes and net charge transfers of the light-evoked bump responses are expected to be overestimated as compared to that of true one-photon responses. This hypothesis is confirm ed by a numerical simulation of light-evoked bump responses using experimentally recorded spontaneous bumps (at times larger than 1 s after the flash) as the simulation material. We show that the superposition of one-photon events in the light-evoked bump responses due to Poisson statistics settles the question why their amplitudes and net charge transfers are found to be larger than that of the spontaneous bumps. We suggest that true one-photon responses evoked by a light flash and spontaneous bumps start from the same activated rhodopsin state and take the same biochemical pathway.

The Sensitivity of the Ventral Nerve Photoreceptor of Limulus Recovers after Light Adaptation in Two Phases of Dark Adaptation

The time course of the recovery of the sensitivity of the Limulus ventral nerve photoreceptor was measured during dark adaptation following light adaptation by a bright 1 or 5 s illumination. The stimulus intensity ICR of a 300 μs light flash evoking a response of criterion amplitude (receptor potential or receptor current under voltage clamp conditions) was used as measure of sensitivity.The time course of dark adaptation shows two phases with time constants in the range of 5-9 s and 300-500 s (15 °C). Only the first of the two phases is significantly changed when the extracel- lular Ca2+-concentration is varied.The power function ICR = a·Io-tDA-b gives a good data fit for each of the two phases of dark adaptation. In the first phase the factor ax and the exponent bx are decreased when the external calcium is lowered from 10 mmol/1 to 250 μmol/1. Conversely a1 and b1 are increased when the Ca2+-concentration is raised to 40 mmol/1. For the second phase neither a2 nor b2 is changed significantly upon the changes in calcium concentration in the same experiments.The two phases of dark adaptation reflect the behaviour of the two components C1 and C2 of the electrical light response (receptor potential or receptor current). Under the conditions described here C, determines the size of the light response during the first phase of dark adaptation whereas C2 mainly influences the size of the response during the second phase.Interpretation: The fast first phase of dark adaptation is determined by the change in intracellu- lar Ca2+-concentration. The slower second phase of dark adaptation is not primarily calcium- controlled.

The Sensitivity Shift Due to Light Adaptation Depending on the Extracellular Calcium Ion Concentration in Limulus Ventral Nerve Photoreceptor

Receptor potentials of Limulus ventral photoreceptors were recorded in two defined states of moderate light- and considerable dark adaptation (LA, DA) by a repeated stimulus sequence consisting of a conditioning 2 s illumination (white light, response saturating intensity) followed by two 10 ms test flashes at fixed intervals evoking LA and DA responses (intensity varied from threshold to saturation of response amplitude). The half saturating intensity I50 was determined from response height vs log stimulus intensity curves for LA and DA, while the photoreceptor was superfused either by reference saline (physiological ion concentrations, including 10 mmol/l Ca2+) or by test salines in which the [Ca2+] was varied between 40 μmol/l and 100 mmol/l. The sensitivity of the dark-adapted receptor does not significantly depend on the [Ca2+]ex, but the sensitivity shift due to LA (measured by /50) is reduced when the [Ca2+]ex is lowered, and augmented when the [Ca2+]ex is increased. Additional reduction of the [Na2+]ex from 463 mmol/l to 46 mmol/l or increase of the [Mg2+]ex from 50 mmol/l to 100 mmol/l does not counteract the effect of lowered [Ca2+]ex on LA. The results confirm the assumption that a transient increase of the intracellular [Ca2+] supplied from extracellular sources during the light response is the main cause for LA This calcium effect on light adaptation is neither characterized by a calcium/sodium antagonism, nor mimicked by magnesium, in contrast to the calcium effect on the gating of the light-activated ion channels.

The Intensity Dependence of the Receptor Potential of the Limulus Ventral Nerve Photoreceptor in Two Defined States of Light-and Dark Adaptation

Receptor potentials of Limulus ventral nerve photoreceptors were evoked in reproducible states of moderate light- and considerable dark adaptation (LA, DA) with light stimulus intensities from threshold to saturation values using a repeated flash sequence. With increasing light intensities latent period and time-to-peak shortened in the state of both LA and DA The decrease-time is prolonged by increasing stimulus intensities (LA and DA) and continues to rise even when the amplitude of the receptor potential is already saturated. The sigmoid response height vs. log stimulus intensity curve is shifted upon LA towards higher stimulus intensities, and its steepness is increased. LA prolongs latent period and time-to-peak at low light intensities; at high light intensities, however, the DA-values are longer. The decreasetime of the receptor potential is always shorter in the light-adapted state. The results are discussed according to the assumption that size and duration of the receptor potential are primarily determined by the distribution of bum p latencies. Changes of the receptor potential parameters are explained by changes of bum p size and of bum p latency distribution due to light adaptation.

The Influence of the Extracellular Concentration of Calcium, Magnesium and Sodium on the Repolarizing Phase of the Receptor Potential of the Limulus Ventral Nerve Photoreceptor

1.Lowering the extracellular calcium concentration from 10 mmol/l to 1 nmol/l causes, besides reducing membrane potential (PMP) and peak amplitude (hmax) of the light response of the Limulus ventral nerve photoreceptor (see Stieve and Bruns [1]), a prolongation of the time course of the light response. The retarded time course (characterized by latent-period tlat, time-to-peak tmax, decrease time t2 and decline quotient QHN) caused by low calcium concentration is not antagonized by either reducing the sodium concentration (from 0.5 to 0.05 mol/l) or increasing the magnesium concentration (from 5× 10-5 to 5 × 10-2 mol/l) in contrast to the effects on the PMP and hmax. 2.This effect of lowering the calcium concentration on the time course of the ReP is distinctly different from that on membrane potential and transient of the ReP described before. It is not characterized by a calcium/sodium binding competition but is probably more closely related to the bump-generating mechanism. It can be explained on the assumption that the time parameters of the ReP are primarily determined by the latency distribution of the underlying bumps which is expanded under low calcium conditions.