Takako Nishi

Roles of cyclic GMP and inositol triphosphate in phototransduction of the molluscan extraocular photoreceptor

The internal messengers mediating the photocurrent of the molluscan extraocular photoreceptor, A-P-1, were examined. In the dark, pressure-injection of cGMP into the A-P-1, voltage-clamped at resting levels, produced a rapid outward current, associated with an increase in conductance. However, the cGMP-induced current and increase in conductance were suppressed by subsequent photostimulation, suggesting hydrolysis of cGMP by light. The steady-state I/V relation for the cGMP-induced current was non-linear. The I/V relation for the instantaneous cGMP-induced current, measured 50 ms after the beginning of a voltage step, was linear, and reversed at the membrane potential, -67 mV, which corresponded to the K+ equilibrium potential of A-P-1 in 10 mM K+ normal saline. These findings indicate that the internal cGMP induces a voltage- and time-dependent K+ current. Since the photocurrent results from the suppression of a voltage- and time-dependent K+ current similar to above, the photocurrent is considered to be equivalent to the suppression of the cGMP-induced current. Short pressure-injection of GDP-beta-S into A-P-1 reduced the subsequent photocurrent. The photocurrent was also suppressed after an external application of Pertussis toxin. On the other hand, the photocurrent was amplified by prior pressure-injection of inositol 1,4,5-trisphosphate (IP3). However, a short pressure-injection of neomycin into A-P-1 depressed the subsequent photocurrent. These results suggested that the cGMP-induced (dark) current is mediated by cGMP, and that hydrolysis of cGMP by light leads to the photocurrent, then being modified by another messenger, IP3, to be amplified.(ABSTRACT TRUNCATED AT 250 WORDS)

Single K+ channels closed by light and opened by cyclic GMP in molluscan extra-ocular photoreceptor cells ☆

We report the first recordings of the light-sensitive channel which is active during dark and is closed by light in the Onchidium extra-ocular photoreceptor cells. This light-sensitive channel was K-selective and was not blocked by extracellular Ca2+ and Mg2+. Application of cyclic GMP to excised inside-out patches activated (opened) a channel that appeared to be the same as the light-sensitive channel recorded from the same membrane in the intact cell.

Light-dependent K+ Channels in the Mollusc Onchidium Simple Photoreceptors Are Opened by cGMP

Light-dependent K+ channels underlying a hyperpolarizing response of one extraocular (simple) photoreceptor, Ip-2 cell, in the marine mollusc Onchidium ganglion were examined using cell-attached and inside-out patch-clamp techniques. A previous report (Gotow, T., T. Nishi, and H. Kijima. 1994. Brain Res. 662:268–272) showed that a depolarizing response of the other simple photoreceptor, A-P-1 cell, results from closing of the light-dependent K+ channels that are activated by cGMP. In the cell-attached patch recordings of Ip-2 cells, external artificial seawater (ASW) was replaced with a modified ASW containing 150 mM K+ and 200 mM Mg2+ to suppress any synaptic input and to maintain the membrane potential constant. When Ip-2 cells were equilibrated with this modified ASW, the internal K+ concentration was estimated to be 260 mM. Light-dependent single-channels in the cell-attached patch on these cells were opened by light but scarcely by voltage. After confirming the light-dependent channel activity in the cell-attached patches, an application of cGMP to the excised inside-out patches newly activated a channel that disappeared on removal of cGMP. Open and closed time distributions of this cGMP-activated channel could be described by the sum of two exponents with time constants τo1, τo2 and τc1, τc2, respectively, similar to those of the light-dependent channel. In both the channels, τo1 and τo2 in ms ranges were similar to each other, although τc2 over tens of millisecond ranges was different. τo1, τo2, and the mean open time τo were both independent of light intensity, cGMP concentration, and voltage. In both channels, the open probability increased as the membrane was depolarized, without changing any of τo2 or τo. In both, the reversal potentials using 200- and 450-mM K+-filled pipettes were close to the K+ equilibrium potentials, suggesting that both the channels are primarily K+ selective. Both the mean values of the channel conductance were estimated to be the same at 62 and 91 pS in 200- and 450-mM K+ pipettes at nearly 0 mV, respectively. Combining these findings with those in the above former report, it is concluded that cGMP is a second messenger which opens the light-dependent K+ channel of Ip-2 to cause hyperpolarization, and that the channel is the same as that of A-P-1 closed by light.

A light-induced decrease of cyclic GMP is involved in the photoresponse of molluscan extraocular photoreceptors

The depolarizing photoreceptor potential in the molluscan extraocular photoreceptor, A-P-1, results from the light-induced suppression of specific K+ currents. An application of cGMP or IBMX (inhibitor of phosphodiesterase) to A-P-1-evoked light-suppressed currents, similar to the above specific K+ currents. Thus, the light-induced decrease of cGMP levels in A-P-1 may be responsible for the photoreceptor potential, like vertebrate phototransductions.

Mechanisms of hyperpolarization induced by two cytokines, hTNFα and hIL-1α in neurons of the mollusc,Onchidium

Abstract The voltage and current responses induced by extracellular tumor necrosis factor (hTNFα) or interleukin-1 (hIL-1α) on the Be-1 and Es-1 neurons of theOnchidium ganglia were examined. Pressure-ejected hTNFα or hIL-1α produced an inhibitory, hyperpolarized effect in unclamped neurons. In the same neurons voltage-clamped at their resting potential levels, the same hTNFα or hIL-1α elicited an outward current having a time course similar to that of the hyperpolarization, associated with a decreased membrane conductance. The hTNFα- or hIL-1α-induced outward current did not reverse even at positive membrane potentials considerably above +100 mV in the absence of ouabain (a specific blocker of Na-pump). In the presence of ouabain, the hTNFα- or hIL-1α-induced current was reduced over a wide range of membrane potential, so that the current reversed at about +20 mV. Lowering the external Na+ concentration from 450 to 200 mM in the presence of ouabain, shifted the reversal potential from +20 to 0 mV, to near the shift value of 20.8 mV predicted by the Nernst equation for a Na+-selective conductance. Neither an increase nor a decrease of extracellular K+, Cl− or Ca2+, however, significantly altered the current induced by hTNFα or hIL-1α. These suggest that the hTNFα- or hIL-1α-induced hyperpolarization or outward current response is mediated by two mechanisms, a decrease in Na+ conductance and activation of the Na-pump.

The light-suppressible K+ conductance and evaluation of internal messenger candidates in the molluscan extraocular photoreceptor.

A photoreceptor potential produced by a decrease in membrane conductance was not thought to occur in any invertebrate photoreceptors. However, we have found that the molluscan extraocular photoreceptor, A-P-1 responds to light with a depolarizing receptor potential due to a decrease in K+ conductance, so that the photoresponse associated with a decrease in membrane conductance is not unique to the vertebrate photoreceptor. The properties that the light-suppressible K+ conductance is time- and voltage-dependent are explained by comparison with those of the single channel conductance obtained in patch-clamp of both vertebrate and invertebrate photoreceptors. The noise analysis of the light-induced current suggest that this macroscopic light-suppressible conductance consists of channels. It is concluded that the light-suppressible K+ conductance is mediated by hydrolysis of cGMP which reduces internal cGMP, in agreement with the cGMP hypothesis of vertebrate phototransduction and that the hydrolysis may be modified by IP3.