Phe-Met-Arg-Phe-amide Activates a Novel Voltage-dependent K⁺ Current through a Lipoxygenase Pathway in Molluscan Neurones

The neuropeptide Phe-Met-Arg-Phe-amide (FMRFa) dose dependently (ED₅₀ = 23 nM) activated a K⁺ current in the peptidergic caudodorsal neurones that regulate egg laying in the mollusc Lymnaea stagnalis. Under standard conditions ([K⁺]_o = 1.7 mM), only outward current responses occurred. In high K⁺ salines ([K⁺]_o = 20 or 57 mM), current reversal occurred close to the theoretical reversal potential for K⁺. In both salines, no responses were measured below −120 mV. Between −120 mV and the K⁺ reversal potential, currents were inward with maximal amplitudes at ∼−60 mV. Thus, U-shaped current–voltage relations were obtained, implying that the response is voltage dependent. The conductance depended both on membrane potential and extracellular K⁺ concentration. The voltage sensitivity was characterized by an e-fold change in conductance per ∼14 mV at all [K⁺]_o. Since this result was also obtained in nearly symmetrical K⁺ conditions, it is concluded that channel gating is voltage dependent. In addition, outward rectification occurs in asymmetric K⁺ concentrations. Onset kinetics of the response were slow (rise time ∼650 ms at −40 mV). However, when FMRFa was applied while holding the cell at −120 mV, to prevent activation of the current but allow activation of the signal transduction pathway, a subsequent step to −40 mV revealed a much more rapid current onset. Thus, onset kinetics are largely determined by steps preceding channel activation. With FMRFa applied at −120 mV, the time constant of activation during the subsequent test pulse decreased from ∼36 ms at −60 mV to ∼13 ms at −30 mV, confirming that channel opening is voltage dependent. The current inactivated voltage dependently. The rate and degree of inactivation progressively increased from −120 to −50 mV. The current is blocked by internal tetraethylammonium and by bath- applied 4-aminopyridine, tetraethylammonium, Ba²⁺, and, partially, Cd²⁺ and Cs⁺. The response to FMRFa was affected by intracellular GTPγS. The response was inhibited by blockers of phospholipase A₂ and lipoxygenases, but not by a cyclo-oxygenase blocker. Bath-applied arachidonic acid induced a slow outward current and occluded the response to FMRFa. These results suggest that the FMRFa receptor couples via a G-protein to the lipoxygenase pathway of arachidonic acid metabolism. The biophysical and pharmacological properties of this transmitter operated, but voltage-dependent K⁺ current distinguish it from other receptor-driven K⁺ currents such as the S-current- and G-protein-dependent inward rectifiers.