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Inward currents activated by 8-bromc-cGMP and by muscarinic agonist were compared in N1E-115 mouse neuroblastoma cells using perforated- patch voltage clamp and Fura-2 imaging. The cGMP analog activates a voltage-independent inward current that is carried at least in part by Ca2+ because it persists in Na(+)-free saline when Ca2+ is present and is blocked by external Mn2+ and Ba2+. The current is similar to the inward current that develops during stimulation of M1 muscarinic receptors, and the currents activated by agonist and by 8-bromo-cGMP are not additive, indicating that the same pathway is involved. Inhibition of cGMP production with NG-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide (NO)-synthase, prevents activation of Ca2+ current by agonist without affecting the content of intracellular Ca2+ stores or the ability of agonist to mobilize Ca2+. The inhibition is overcome by 8-bromo-cGMP. LY83583, a competitive inhibitor of guanylyl cyclase, reversibly blocks activation of Ca2+ current by agonist, again without affecting the content of Ca2+ stores or Ca2+ release. Rp-8-pCPT-cGMPS, an inhibitory analog of cGMP, also reduces the Ca2+ current and reduces Ca2+ influx during muscarinic activation. It is concluded that cGMP is the necessary and sufficient intermediate in the pathway linking muscarinic receptor occupancy to the activation of voltage-independent Ca2+ current. The pathway involves positive feedback. Calcium entering via voltage-independent channels preferentially stimulates NO-synthase, which leads to enhanced cGMP production and greater Ca2+ influx. Positive feedback may explain the rapid increase in cGMP that occurs during muscarinic receptor activation.

The activation of Ca-dependent K+ current, Ic, was studied in macropatches on the cell bodies of molluscan neurons. When a depolarizing voltage-clamp pulse was applied repeatedly, Ic facilitated in a manner that resembled the facilitation of synaptic transmitter release. Facilitation was characterized by an increase in Ic amplitude, a progressive increase in instantaneous outward current, and a decrease in utilization time. Experiments were done to investigate the mechanism responsible for Ic facilitation. Facilitation was reduced by microinjection of an exogenous Ca2+ buffer into the cytoplasm, indicating that facilitation is a Ca(2+)-dependent process. It was also reduced at elevated temperatures. Conversely, facilitation was greatly potentiated by blocking the Na/Ca exchange mechanism. It is concluded that the facilitation of Ca-dependent K+ current results from the accumulation of Ca2+ at the inner face of the membrane during the repeated activation of Ca2+ channels by depolarization. The Ca2+ indicator fluo-3 was used in fluorescence imaging experiments to measure changes in [Ca]i near the cell membrane during repeated depolarizing pulses and the interpretation of these results was aided by numerical simulations of Ca2+ accumulation, diffusion, and buffering in the peripheral cytoplasm. These experiments showed that the time course of Ic facilitation matches the time course of Ca2+ accumulation at the membrane. It was found that the strength of Ic facilitation varies among patches on the same neuron, suggesting that the accumulation of Ca2+ is not uniform along the inner surface of the membrane and that gradients in [Ca]i develop and are maintained during trains of depolarizing pulses. Potential mechanisms that may lead to local differences in Ca2+ accumulation and Ic facilitation are discussed.

The response of Aplysia abdominal ganglion neuron L2 to the molluscan neuroactive peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) was studied in voltage-clamp experiments. In all of the experiments, focal application of the peptide to the soma activated an inward rectifier current and reduced the apparent amplitude of the transient K current, IA. In a few cells, Na and K currents were activated in addition to these effects. Voltage-jump experiments were performed to study the ionic dependence, kinetics, and voltage dependence of the inward rectifier. Inward rectification increased exponentially during hyperpolarizing pulses and recovered exponentially on return to the resting potential. The reversal potential was variable, but was near -40 mV at the beginning of experiments. Inward rectification was insensitive to changes in external Na, Ca, or K concentration, but lowering the external Cl concentration had complicated effects on current amplitude. When KCl microelectrodes were used, perfusion with low-Cl external saline increased the amplitude of the peptide-dependent inward rectifier and shifted its reversal potential to a more positive voltage. With KAc microelectrodes, perfusion with low-Cl saline reduced the amplitude of the current. Inward rectification increased when a KAc microelectrode was withdrawn and replaced with a low-resistance KCl electrode, even when there was no measurable change in reversal potential. These results suggest that the FMRFamide-dependent inward rectifier is a Cl current that, like the current described by Chesnoy-Marchais (1982, 1983), is modulated by intracellular Cl. FMRFamide reduced the apparent amplitude of IA without affecting the voltage dependence of IA activation or inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bursting pacemaker neurons of Tritonia and Aplysia were studied in voltage-clamp experiments to determine the ionic requirements of the slow outward tail current. This current is important for terminating bursts and hyperpolarizing the neuron during the interval between bursts and therefore contributes to the timing of pacemaker activity. A method for rapid extracellular perfusion of the neuron soma with solutions of different ionic composition was used to study the ionic dependence of the tail current. Measurements of the current-voltage relationship were made at different times during the decay of the tail current to determine the reversal potential in different ionic solutions. These experiments showed that 2 or more slow outward currents contribute to the tail current. Temperature had a large effect on these currents. At cold temperatures (10–15 degrees C), the tail current was predominantly a K current. At warm temperatures (20–23 degrees C), both a K current and a K-insensitive outward current were seen. Bursting pacemaker activity occurred throughout this temperature range. Both the K current and the K-insensitive current required Ca influx for activation. These findings help to reconcile conflicting reports in the literature concerning the ionic dependence of the slow outward tail current and suggest a mechanism for temperature compensation of bursting activity in these pacemaker cells.

The spatial distribution of Ca current in molluscan neuron cell bodies was studied using a large patch method in combination with 2- microelectrode voltage clamp. The method has a spatial resolution equal to about 0.1% of the cell body area. Ca current is not uniformly distributed. The current density varies between patches, changing by as much as a factor of 2.5 over a distance of 20 micron, and there is evidence that Ca current occurs in “hot spots” involving a few hundred channels. The current density increases in a moderately steep gradient from the soma cap, opposite the axon, toward the axon hillock. Ca currents in patches from different regions of the soma are qualitatively different. Currents near the soma cap do not inactivate or inactivate weakly during depolarization, while currents of equal density nearer the axon hillock exhibit pronounced inactivation. The strength of inactivation increases in parallel with the gradient in current density, but local differences in current density, or in the number of active Ca channels, do not explain the variability in inactivation. Inactivating and noninactivating Ca currents could not be distinguished on the basis of activation or deactivation kinetics, voltage dependence of activation, or sensitivity to hyperpolarizing conditioning pulses. Also, the amplitude of noninactivating current near the soma cap is reduced by intracellular Ca injection showing that, like the whole-cell current, Ca current in this region is subject to Ca-dependent inactivation. The data favor the hypothesis that these cells express only one type of Ca channel. Differences in the strength of inactivation may result from local differences in cytoplasmic Ca buffering, local modification of Ca channels in a way that changes their sensitivity to Ca-dependent inactivation, or local differences in the availability of cytoplasmic factors or enzymes that are necessary for inactivation.