Pharmacological Investigation of Protein Kinase C- and cGMP-Dependent Ion Channels in Cultured Olfactory Receptor Neurons of the Hawkmoth Manduca sexta

Abstract

In the hawkmoth Manduca sexta, pheromone stimuli of different strength and duration rise the intracellular Ca2+ concentration in olfactory receptor neurons (ORNs). While second-long pheromone stimuli activate protein kinase C (PKC), which apparently underlies processes of short-term adaptation, minute-long pheromone stimuli elevate cyclic guanosine monophosphate (cGMP) concentrations, which correlates with time courses of long-term adaptation. To identify ion channels involved in the sliding adjustment of olfactory sensitivity, inside-out patch clamp recordings on cultured ORNs of M. sexta were performed to characterize Ca2+-, PKC-, and cGMP-dependent ion channels. Stepping to positive holding potentials in high intracellular Ca2+ elicits different Ca2+-dependent ion channels, namely small-conductance channels (2–20 ps), medium-conductance channels (20–100 ps), and large-conductance channels (>100 ps). Ion channels of 40, 60, and 70 ps opened after PKC activation, whereas 10- and >100-ps channels were observed less frequently. Application of 8-bromo cyclic guanosine monophosphate opened 55- and 70-ps channels and increased the open probability of >100-ps channels, whereas even in the presence of phorbol ester 40-ps channels were inhibited. Thus, cGMP elevations activate a different set of ion channels as compared with PKC and suppress at least one PKC-dependent ion channel.

Deterrence of feeding in Rhodnius prolixus (Hemiptera: Reduviidae) after treatment of antennae with a nitric oxide donor

“…There is evidence that NO increases cGMP concentration (Stengl et al, 2001) and activates ion channels in the ORNs of M. sexta (Zufall & Hatt, 1991;Dolzer et al, 2008). Stengl & Zintl (1996) found NADPH diaphorase activity (indicative of NO synthase activity) in ORNs and olfactory supporting cells of M. sexta.…”

“…Most studies on NO and the sensorial function in insects have used biochemical (Stengl & Zintl, 1996;Redkozubov, 2000;Newland & Yates, 2008) or electrophysiological (Stengl et al, 2001;Waserman & Itagaki, 2003;Dolzer et al, 2008) methods. However, the insect's behavioural response was only studied in a few cases, mainly in the locust (Newland & Yates, 2007;Wenzel et al, 2005).…”

Perfusion with cAMP analogue affects pheromone-sensitive trichoid sensilla of the hawkmothManduca sextain a time-dependent manner

“…An apparently directly cAMPdependent non-specific cation channel and a cAMP-dependent Ca 2+ channel were identified in M. sexta , next to other cyclic nucleotide-sensitive cation and potassium channels (Dolzer, 2002;Dolzer et al, 2008). While these channels were specifically sensitive to 8bcAMP at concentrations lower than 1 mmol l -1 cyclic nucleotide, at considerably higher concentrations of cAMP cGMP-dependent cation channels were also activated.…”

“…Recently it was shown that heterologously expressed insect olfactory receptors form cAMP-sensitive ligand-gated ion channel complexes, with the highly conserved chaperone protein OR83b (Sato et al, 2008;Wicher et al, 2008). Furthermore, cyclic nucleotide-sensitive ion channels were characterized in patch clamp recordings of cultured ORNs of Manduca sexta (Dolzer et al, 2008;Krannich and Stengl, 2008). Preliminary biochemical experiments indicate that cyclic nucleotide concentrations express time-dependent rhythms in insect antennae (W. Peters, T. Schendzielorz and M.S., unpublished).…”

Identification and characterization of the bombykal receptor in the hawkmoth Manduca sexta

“…Next, a possible dependence of the BAL response on PLC or PKC activity was examined, as reported previously for hawkmoth pheromone transduction (Stengl, 1993(Stengl, , 1994(Stengl, , 2010Dolzer et al, 2008) and for fruitfly Orco (Sargsyan et al, 2011;Getahun et al, 2013). Thus, Ca 2+ imaging experiments were performed in HEK293 cells expressing MsexOrco and MsexOr1.…”