Ionic mechanism of the outward current induced by intracellular injection of inositol trisphosphate into Aplysia neurons

Abstract

Inositol 1,4,5-trisphosphate (InsP3) has been proposed to be the intracellular second messenger in the mobilization of Ca2+ from intracellular stores in a variety of cell types. The ionic mechanism of the effect of intracellularly injected InsP3 on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure-injection, and ion-substitution techniques. Brief pressure injection of InsP3 into a neuron voltage- clamped at -40 mV reproducibly induced an outward current (10–60 sec in duration, 20–60 nA in amplitude) associated with a conductance increase. The current was increased by depolarization and decreased by hyperpolarization up to -80 mV, where it disappeared. Extracellular application of tetraethylammonium (TEA; 5 mM) blocked the InsP3-induced outward current, and the current was not affected by the presence of bath-applied 4-aminopyridine (4-AP; 5 mM). The InsP3-induced outward current recorded at a holding potential of -40 mV increased in amplitude in low-K+ solutions and decreased in amplitude in high-K+ solutions. Alteration of [Cl-]0, as well as perfusion with Ca2+ free plus 2 mM EGTA solution, did not affect the outward current. The InsP3- induced outward current was found to disappear when the neuron was injected with the Ca2+ chelator EGTA. The outward current evoked by repeated InsP3 injection at low doses exhibited summation and facilitation and, at high doses, was shown to desensitize. The calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W-7; 20–50 microM), inhibited both the InsP3-induced and the Ca2+-activated outward currents. An intracellular pressure injection of Ca2+ ions into the same identified neuron was shown to produce an outward current associated with a K+ conductance increase similar to the InsP3-induced current, and the current was blocked by bath-applied TEA (5mM). These results suggest that brief pressure injection of InsP3 into certain identified neurons of Aplysia induces a 4-AP-resistant, TEA-sensitive K+ current activated by increased intracellular free Ca2+ concentration, and this increase might be the result of the mobilization of Ca2+ from intracellular stores by InsP3.

Effect of intracellular injection of inositol trisphosphate on cytosolic calcium and membrane currents in Aplysia neurons

“…Our results also agree with those of Sawada et al (1987) for the InsP,-activated outward current, and those of Parker and Ivorra (1990b) for chloride currents and Ca, increases.…”

“…In previous studies, the effect of InsP, in nerve cells ofAplysia kurodai (Sawada et al, 1987) and Aplysia calijbrnica (Sholz et al, 1988) was indirectly shown to mobilize Ca*+, by measuring membrane currents. Fink et al (1988) have shown, using digital imaging of fura-fluorescence, that InsP, increases Ca, in Aplysia bag cells.…”

“…Scholz et al (1988) described a similar biphasic response caused by InsP, injections in Aplysia. Sawada et al (1987) found only outward currents in Aplysia kurodai neurons. The biphasic response shown in Figure 6A resembles the response caused by Ca2+ injections in Helix neurons (Hofmeier and Lux,198 1) and could be the Ca2+-dependent, nonspecific cation current responsible for maintaining repetitive spiking during the burst of Helix (Swandulla and Lux, 1985) and Aplysia (Kramer and Zucker, 1985a,b) neurons.…”

Mechanisms underlying intracellular signal transduction of the slow IPSP in submucous neurones of the guinea‐pig caecum.

“…Calmodulin is an intracellular Ca21 receptor protein, and is reported to regulate a variety of intracellular processes (Kennedy, 1989;Schulman & Lou, 1989;Hemmings, Nairn, McGuinness, Huganir & Greengard, 1989). The outward current induced by the intracellular injection of InsP3 has been reported to involve a calmodulin-dependent mechanism which opens a potassium channel in Aplysia neurones (Sawada, Ichinose & Maeno, 1987). It has also been suggested that the release of neurotransmitter may be modulated by a calmodulin kinase-mediated phosphorylation of synapsin I (Schulman & Lou, 1989).…”

myo‐Inositol metabolites as cellular signals

“…Additional neuronal effects of Ins(1,3,4,5,6)P5 and InsPs have recently been described following their microinjection into identified neurons in the abdominal ganglia of the marine mollusc, Aplysia [202,2031. The injection of InsP6, achieving a calculated intracellular concentration of 0.2 pM, evoked a biphasic response consisting of an initial inward current carried mainly by Na+ and Ca2+, followed by a prolonged hyperpolarisation due to stimulation of an outward K + channel.…”