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 approximately -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 approximately 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 approximately 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 approximately 36 ms at -60 mV to approximately 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 GTPgammaS. 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.
Kits
K S
KS
Graduate School Neurosciences Amsterdam, Research Institute of Neuroscience, Vrije Universiteit, Faculty of Biology, 1081 HV Amsterdam, Netherlands. ksk@bio.vu.nl
Lodder
J C
JC
Veerman
M J
MJ
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D004594:Electrophysiology; D019835:FMRFamide / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D007700:Kinetics; D008084:Lipoxygenase / Q000378:metabolism*; D008195:Lymnaea / Q000502:physiology*; D009474:Neurons / Q000378:metabolism*; D009994:Osmolar Concentration; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000502:physiology; D015221:Potassium Channels / Q000378:metabolism / Q000502:physiology*; D015290:Second Messenger Systems / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology
D015221:Potassium Channels; D016718:Arachidonic Acid; D019835:FMRFamide; D008084:Lipoxygenase; D019204:GTP-Binding Proteins; D011188:Potassium