Ionic currents of cultured olfactory receptor neurons from antennae of male manduca sexta

Abstract

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the ionic currents of insect olfactory receptor neurons (ORNs) in vitro. The cells were isolated from the antennae of male Manduca sexta pupae at stages 3-5 of adult development and maintained in primary cell culture. After 2-3 weeks in vitro, the presumptive ORNs had resting potentials of -62 ± 12 mV (n = 18) and expressed at least 1 type of Na+ channel and at least 3 types of K+ channels. Na+ currents, recorded in the whole-cell mode, were reversibly blocked by 0.1 μM tetrodotoxin. The predominant type of K+ channel observed was a voltage-activated K+ channel (γ = 30 pS) with characteristics similar to those of the delayed rectifier. The activity of the 30-pS K+ channel could be inhibited by the application of nucleotides to the cytoplasmic face of inside-out patches of membrane. The nucleotides had relative potencies as follows: ATP > cGMP > cAMP, with an inhibition constant for ATP of K1 = 0.18 mM. Raising the intracellular Ca2+ concentration from 0.1 to 5 μM induced the opening of a Ca2+- activated K+ channel (γ = 66 pS at 0 mV) that had a low voltage sensitivity. A third, transient type of K+ channel (γ = 12-18 pS) could be activated by depolarizing voltage steps from very negative resting potentials. Properties of this channel were similar to those of the "A-channel." These results support the conclusion that M. sexta ORNs differentiate in vitro and provide the basis for studying primary mechanisms of olfactory transduction.