Ventral Nerve Photoreceptor Research Articles

Desensitization of the photoresponse in Limulus ventral photoreceptors by protein kinase C precedes rhabdomere disorganization and endocytosis.

Limulus photoreceptors utilize the phosphoinositide pathway to generate light-induced single photon events (quantum bumps) that sum to form the depolarizing receptor potential. The protein kinase C (PKC) activator, (-)-indolactam V (ILV) rapidly desensitizes the light response in Limulus ventral nerve photoreceptors. Within 10 min of extracellular application, 100 nM (-)-ILV caused a decrease in the mean amplitude of quantum bumps to 38% of control values. PKC activation by (-)-ILV also causes photosensitive membrane disorganization and endocytosis. To investigate whether this precedes desensitization of the electrical response, we fixed cells after 10-min incubation with 25 microM (-)-ILV, a concentration sufficient to cause a 1000-fold desensitization of the receptor potential. The photosensitive microvilli of these photoreceptors remained narrow, densely packed, and well organized. Increasing the incubation time to 60 min did, however, induce disorganization and swelling of the microvilli and endocytosis of the photosensitive membrane, as previously reported. Measurement of membrane capacitance did not indicate a significant reduction in membrane area accompanying desensitization by (-)-ILV. PKC-induced reduction in light sensitivity therefore precedes the detection of ultrastructural changes in the rhabdomeral membrane and is not due to a net loss of membrane.

Putative inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in Limulus photoreceptors

Invertebrate microvillar photoreceptors utilize the phosphoinositide cascade to transduce light stimuli [24]and inositol 1,4,5-trisphosphate is thought to be one of the messengers that triggers the electrical response by mobilizing intracellular stored calcium. [14]To further characterize the role of the phosphoinositide signaling pathway in invertebrate phototransduction, we have examined the distribution of inositol 1,4,5-trisphosphate receptors in Limulus lateral eye and ventral nerve photoreceptors using an immunohistochemical approach combined with confocal microphotolysis of caged inositol 1,4,5-trisphosphate. We have localized the inositol 1,4,5-trisphosphate receptor using an antibody raised against a highly conserved region of the N-terminal of the protein. In lateral eye photoreceptors, the antibody intensely stains cytoplasm directly beneath the photoreceptive microvilli, containing subrhabdomeral cisternae of endoplasmic reticulum. In ventral nerve photoreceptors, the distribution of immunostaining was more homogeneous than within the lateral eye photoreceptors. Simultaneous confocal microphotolysis of caged inositol 1,4,5-trisphosphate and Ca 2+ measurements using the fluorescent indicator Calcium Green 5N were performed to estimate inositol 1,4,5-trisphosphate-induced Ca 2+ release in functionally distinct areas of the ventral nerve photoreceptors. This is the first direct demonstration of the localization of putative inositol 1,4,5-trisphosphate receptor in invertebrate visual cells. The inositol 1,4,5-trisphosphate receptor appears to be localized predominantly to endoplasmic reticulum and taken in conjunction with earlier physiological data from other workers, our result supports a central role for the phosphoinositide pathway in visual transduction in Limulus photoreceptors.

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 are activated by pathways operating independently of phospholipase C.

Cytosolic Calcium Transient and Light Response in Limulus Ventral Nerve Photoreceptors: Supralinearity in Response-Intensity Curves

Abstract Light-evoked intracellular electrical responses were measured simultaneously with the changes in intracellular free Ca2+ or Sr2+ concentration by means of arsenazo III. The response-versus-stimulus intensity characteristics was measured in two states of relative light adaptation in strontium-containing saline (10 mᴍ Sr2+; 0.25 mᴍ Ca2+). Supralinear parts of the curves with slopes > 1 were found for the receptor current amplitude and the current­-time integral as well as for the amplitude of the arsenazo signal within at least 0.3 log units of identical stimulus intensity range. Maximum slopes of response-versus-intensity curves are on average approximately 1.7 for the receptor current and the arsenazo signal in both adaptational states. The rise in time of the arsenazo signal evoked by a light flash shows two phases. In strontium saline we observe a fast increase at the onset of the arsenazo signal which is detectable within the first 20-100 ms of the signal rise and a slow further increase to the maximum, with a duration of 0.7-6 s. In the detailed analysis of both phases we observed that the amplitude of each phase increases supralinear, i.e. disproportionate to the stimulus intensity. The fast rising phase of the arsenazo signal is not visibly voltage-dependent. This fast rising phase might be a monitor of an IP3-sensitive calcium or strontium release. Since the amplitude of the fast rising phase increases supralinear with the stimulus intensity, we propose the interpretation that more than one IP3-molecule must bind to the IP3-receptor to open this channel of the intracellular calcium store. Only the slow rising phase is voltage-dependent. The kinetic of this slow rising phase was accelerated with more negative membrane potentials. This predominant slow phase correlates with the receptor current-time integral. Therefore the slow rising phase of the arsenazo signal might be a monitor for influx of calcium or strontium through light activated ion channels in the plasma membrane. The supralinearity of the amplitude of the slow rising phase might indicate the supralinear increase in calcium or strontium, which is carried by the receptor current. Additionally, we calculated the fraction of strontium and calcium ions of the total receptor current. When calcium was replaced by strontium in the superfusate about 3-30% of the receptor current are carried by strontium and calcium ions. In physiological saline calcium contributes about 1-1.5% to the receptor current.

Immunological demonstration of Gq-protein in Limulus photoreceptors.

The phototransduction cascade in invertebrates involves the coupling of rhodopsin activation to the action of the enzyme phospholipase C. This step is performed by G-proteins. An antibody against the alpha-subunit of a mouse Gq type G-protein recognized protein bands in Western blots of lateral eye and ventral nerve photoreceptors of Limulus. The protein bands had an apparent molecular mass of about 42 kDa. The antibody also recognized protein bands of a similar molecular mass in immunoblots of brain and intestine tissue. Immunoreactivity was found in lateral eye frozen sections where it was confined to the rhabdom region. When the antibody was applied to ultrathin sections of ventral nerve photoreceptors, the highest density of labeling was found on the rhabdomeral microvilli, but gold particles were also scattered in the cytoplasm. We conclude that a G-protein of the type Gq participates in the phototransduction of Limulus.

A mathematical model for the components of the receptor current in Limulus ventral nerve photoreceptors

The light-activated current in the ventral photoreceptor of Limulus consists of three components. The components have different physical and biochemical properties indicating the operation of different transduction pathways. The second current component is activated via the phospholipase C pathway, which releases calcium from intracellular stores by inositol trisphosphate. The channels of the other two components are probably gated by cyclic nucleotides of different types. We demonstrate here a mathematical model which describes the current components. Current components were fitted by a function consisting of the Fermi function and a single exponential function, describing the activation and deactivation, respectively. The model has four parameters for each current component: (1) a scaling factor for the amplitude; (2) the half-time of activation; (3) the time constant of activation; (4) the time constant of deactivation. The light-intensity dependences of these parameters are different for the three components, reflecting different underlying enzyme reactions. The model makes the quantitative analysis of the current components under different conditions possible.

Receptor current oscillations evoked by local illumination in Limulus ventral nerve photoreceptors

Light flashes to spots on ventral photoreceptors induced complex current bursts or oscillations. The number of peaks in a burst was not correlated with the number of effective photons. Intervals between peaks were constant in each cell. Net charge fluxes during bursts were larger than the charge flux during a single bump multiplied by the number of peaks. The number of peaks, current-time-integrals, and kinetics of bursts evoked either by light spots or by illumination of the same entire cell were similar. Occasionally, bursts appeared at stimulus intensities corresponding with only 1–2 effective photons. At higher intensities they occurred more frequently, and the average burst-evoking intensity corresponded with 6 effective photons. We conclude that a single photoisomerization can induce a current burst due to a local oscillatory process.

Differential calcium sensitivity of two types of quantum bumps in Limulus ventral nerve photoreceptors

Ventral photoreceptors in Limulus polyphemus were continuously illuminated with dim light and produced two types of quantum bumps. Bumps of one type had a relatively symmetrical shape and fast rise and decay kinetics. In addition, bumps were detected with a small amplitude and slower kinetics. We termed these bump types “C2” and “C1 bumps”, respectively. The half width of a bump divided by its amplitude was found to be a reliable criterion to distinguish between the bump types. This shape quotient is smaller than 0.5 ms/pA for the C2 bumps and larger than 1 ms/pA for the C1 bumps. Lowering the extracellular calcium concentration from 10 mM to 0.25 mM caused an increase in the average amplitude of C2 bumps to 196%. After the injection of small amounts of 1.2 mM EGTA solution the amplitude of these bumps was reduced to about 30 and 50% in two cells studied. By contrast, in both cases C1 bump amplitudes were not affected significantly. We conclude that the two bump types are generated in one photoreceptor by the activation of different transduction pathways. These pathways are differentially sensitive to changes of the cytosolic calcium concentration.

The Light-Stimulated Cytosolic Calcium Transient in Limulus Ventral Nerve Photoreceptors: Two Components in the Rising Phase

1. Light-evoked electrical responses were measured in Limulus ventral nerve photoreceptors simultaneously with changes in the cytosolic free calcium concentration, by means of arsenazo III. 2. It has been shown here for the first time that the rise of the arsenazo signal consists of two phases. Only the slow phase in the rise of the signal depends on the membrane voltage. The reversal potential of the amplitude of this slow rising phase was about +196 mV. After removal of external calcium the reversal potential was about +20 mV. 3. When Na+ in the superfusate was replaced by Li+, the amplitude of the fast rising phase was reduced on the average to 50%, while the slow rising phase was not affected. 4. We conclude that the fast rising phase is caused by release of calcium from internal stores while the slow increase in [Ca2+]i is due to influx across the plasma membrane, possibly through light-activated ion channels.

Selective inhibition of the phospholipase C pathway blocks one light-activated current component in Limulus photoreceptor

Light-activated receptor currents were measured in Limulus ventral nerve photoreceptors by a two electrode voltage clamp. Short flashes stimulate three different current components (C1, C2, C3). Currents were measured before and after injection of neomycin, heparin and BAPTA. These substances inhibit the inositol phosphate pathway at different stages. Each substance selectively blocked the C2 component. After the block of C2 the other two components could be further stimulated, but their amplitudes were usually reduced. High concentrations of BAPTA, however, increased the amplitude (two-fold) and decay time constant (eight-fold) of C1, suggesting that calcium is necessary for the deactivation of the C1 current.

Measurement of cytosolic Ca2+ concentration in Limulus ventral photoreceptors using fluorescent dyes.

Several Ca-sensitive fluorescent dyes (fura-2, mag-fura-2 and Calcium Green-5N) were used to measure intracellular calcium ion concentration, Cai, accompanying light-induced excitation of Limulus ventral nerve photoreceptors. A ratiometric procedure was developed for quantification of Calcium Green-5N fluorescence. A mixture of Calcium Green-5N and a Ca-insensitive dye, ANTS, was injected in the cell and the fluorescence intensities of both dyes were used to calculate the spatial average of Cai within the light-sensitive R lobe of the photoreceptor. In dark-adapted photoreceptors, the initial Cai was 0.40 +/- 0.22 microM (SD, n = 7) as measured with fura-2. Cai peaked in the light-sensitive R lobe at 700-900 ms after the onset of an intense measuring light step, when the spatial average of Cai within the R lobe reached 68 +/- 14 and 62 +/- 37 microM (SD, n = 5) as measured with mag-fura-2 and Calcium Green-5N, respectively. The rate of Cai rise was calculated to be approximately 350 microM/s under the measuring conditions. The resting level of Mg2+ was estimated to be 1.9 +/- 0.9 mM, calculated from mag-fura-2 measurements. To investigate the effect of adapting light on the initial Cai level in the R lobe, a 1-min step of 420 nm background light was applied before each measurement. The first significant (P < 0.05) change in the initial level of Cai occurred even at the lowest adapting light intensity, which delivered approximately 3 x 10(3) effective photons/s. The relative sensitivity of the light-adapted photoreceptors was linearly related to the relative Cai on a double log plot with slope between -4.3 and -5.3. We were unable to detect a Cai rise preceding the light-activated receptor potential. The Cai rise, measured with Calcium Green-5N, lagged 14 +/- 5 ms (SD, n = 32) behind the onset of the receptor potential at room temperature in normal ASW. In the absence of extracellular Ca2+ and at 10 degrees C, this lag increased to 44 +/- 12 ms (SD, n = 17).

Inhibition of the first component of the receptor current in Limulus photoreceptor.

The effect of l-cis-diltiazem on the light-activated current was investigated in Limulus ventral nerve photoreceptor. Intracellularly injected diltiazem blocked the first component, reduced the second one to about 50%, but hardly changed the third component of the receptor current. Extracellularly applied diltiazem elicited a similar effect, however, only after about 60 min incubation suggesting that the substance must diffuse into the cell; probably though the cut end of the axon, to be effective. Results suggest that the ion channels underlying the first component are similar to the cGMP-gated channels of rods and thus can be moderately permeable to calcium ions. This calcium influx is sufficient to regulate the gain of the second component which is activated by the inositol phosphate cycle.

Two components of the receptor current are developed from distinct elementary signals in Limulus ventral nerve photoreceptor

Transient elementary currents, bumps, stimulated by short dim light flashes were measured in ventral nerve photoreceptors of Limulus. It is demonstrated that light activates two types of bumps, which form two distinct components of the receptor current at higher light intensities. The two bump types, which are both assumed to be activated by single absorbed photons, differ in current amplitude and kinetic parameters. The current amplitude of one bump type is smaller than 0.3 nA and that of the other type is in the usual current range of up to several nanoamperes. The average latency of small bumps measured from the short stimulus flash is shorter than that of the large bumps. The small bumps have slower activation kinetics than the large bumps. It is demonstrated that with increasing flash intensity the small bumps overlap first and form a macroscopic current, on top of which the large bumps are superimposed. Results indicate that a single absorbed photon selectively activates only one kind of the enzyme cascades evoking one bump type. We conclude that the active meta conformation of a rhodopsin molecule selectively binds a specific type of G-protein, which is involved in the stimulation of one of the transduction cascades. The two bump types, which are the elements of two macroscopic current components support the previous assumption that light activates different transduction mechanisms in Limulus photoreceptors.

Cyclic nucleotides and inositol trisphosphate activate different components of the receptor current in Limulus ventral nerve photoreceptors

Receptor currents activated by short flashes were measured in ventral nerve photoreceptors of Limulus before and after injection of neomycin or phosphodiesterase (PDE). Neomycin inhibits the inositol cascade and PDE hydrolyses cyclic nucleotides. The effect of these substances was studied on the three current components, which were suggested to be activated by distinct transduction mechanisms. The second component of the current was selectively blocked by neomycin and the third component by PDE. The first component was inhibited by both substances. Thus, two transduction mechanisms for two current components could be unequivocally identified. Results support the previous assumption that light activates distinct mechanisms and transmitters in Limulus photoreceptors.

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 4A) against the G alpha subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with G alpha in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADP-ribosyltransferase C3 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 GDP-beta-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.

Non-independent quantum bumps in Limulus ventral nerve photoreceptors - A new insight in the light transduction mechanism

Single photon-induced transient currents, called quantum bumps were stimulated by short flashes in dark-adapted ventral nerve photoreceptors of Limulus. Flash intensities were set to activate 3 or more bumps. In most cases, current bumps were activated with a constant rate. The frequency of bump occurrence was between 9 and 17 Hz. Results show that consecutive bumps are not independent and that some of them are not activated by a photon. The periodic bump activation indicates a molecular mechanism which quantifies the transmitter release not only by a light quantum, but also by a late phase of the transduction cascade. A model is proposed, in which Ca 2+ ions released from intracellular stores transiently block the further Ca 2+ release by inositol trisphosphate in an all-or-none manner.

Three components in the light-induced current of the Limulus ventral photoreceptor.

1. Light-induced currents were measured in Limulus ventral nerve photoreceptors using a two-electrode voltage clamp. Three kinetically distinct components in the light-induced current could be distinguished by varying the light adaptation state of the photoreceptor and the intensity of the stimulus light. 2. The components could be partly separated by choosing appropriate stimulus intensities and dark adaptation time. Thus the properties of the components could be separately studied. The first component is the first to recover after a light adaptation, appears temporally first in the light-induced response, has the lowest activation threshold and is the smallest. The second component needs a longer time to recover after an adapting illumination and its kinetics differ from that of the other components. Applying a bright stimulus to a dark-adapted cell a third component can be observed late in the response. 3. The time to peak of the first and the third components depended on the stimulus intensity, but not on the dark adaptation time. The time to peak of the second component became shorter the longer the dark adaptation time. For a constant adaptation state the time to the maximum of component 2 was independent, but those of components 1 and 3 were dependent on the membrane voltage. 4. To exclude the possibility of the contribution of voltage-gated currents, light-activated currents were measured at clamp potentials more negative than -50 mV after adding 4-aminopyridine into the bath solution or injecting tetraethyl-ammonium chloride into the cell. The properties of the three components remained unchanged under these conditions. 5. The I-V curve of the first component was flat at negative membrane potentials and had a strong outward rectification at positive membrane potentials. The I-V curve of component 3 showed a negative resistance at potentials more negative than about -30 mV. In contrast, the I-V curve for the second component was always nearly linear. 6. No membrane potential was found where the light-induced current was zero. Instead, current traces close to the reversal potential showed a complex waveform indicating different reversal potentials for the three components. 7. The results indicate that the current components are caused by three different populations of light-sensitive channels. The different activations, deactivations and recovery kinetics of the components suggest that the three types of channels are activated by distinct intracellular transmitters.

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

Monte-Carlo simulation of an inhomogeneous reaction-diffusion system in the biophysics of receptor cells

The numerical simulation of Markov processes is usually performed by means of the minimal process method or Gillespie algorithm. In reaction-diffusion systems including extremely inhomogeneous situations, the direct application of this algorithm meets with severe difficulties which eventually cause extremely large computing times. We present a modification of the minimal process method which make it applicable to such situations even on small size computers within moderate computing times. Our modifications include the use of logarithmic classes for transition probabilities in order to increase the acceptance rate of von Neumann rejection methods, a non-local storage of the lattice state, hash tables and dynamical storage management in order to save memory capacity. Our actual example for demonstrating our modified algorithm is signal transduction in biological receptor cells where transmitter molecules are released on a two-dimensional structure (cell membrane) after a quantum reception event, e.g. a photon capture in photoreceptor cells, and interact with ionic transport channels. We find satisfying agreement of our simulation results with the experimental data for the ventral nerve photoreceptor of Limulus.

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.