cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch

Abstract

The cAMP-dependent Na+ current (INa,cAMP) modulates excitability in many molluscan neurons. Rapid activation of INa,cAMP by cyclic nucleotide, its ion dependence, and its blockade by divalent cations resemble cyclic nucleotide-activated cation currents in vertebrate photoreceptors and olfactory receptors, where activation has been found to be independent of kinase activity. We tested the phosphorylation dependence of INa,cAMP in neurons of the feeding and locomotory networks of the predatory marine snail Pleurobranchaea. Identified neurons of pedal and buccal ganglia were axotomized for recording the INa,cAMP response to iontophoretic injection of cAMP under two-electrode voltage clamp. Intracellular injections of specific peptide inhibitor of protein kinase A had no blocking effects on activation of INa,cAMP by iontophoretic injection of cAMP. Inward single-channel currents were activated in excised inside-out patches during exposure to cAMP in salines without added ATP. Sodium was the major current carrying ion. Two distinct types of INa,cAMP channel activity were observed, where opening probability and open times differed, but conductance was similar, 36.7 pS. These observations suggest that INa,cAMP activation occurs by direct binding of cAMP to a regulatory site at the channel, rather than by phosphorylation.

A mouse homolog of dunce, a gene important for learning and memory in drosophila, is preferentially expressed in olfactory receptor neurons

“…Rather, we postulate that mPDE2 may set some of the parameters required for conductance of the depolarizing potential downstream of the primary signal transduction events. Numerous ion channels exist that are directly or indirectly modulated by cyclic nucleotides (Latorre et al, 1991;Bruggemann et al, 1993;Sudlow et al, 1993); in fact, the cyclic nucleotide-gated cation channel present in cilia has also been found in the dendrite and soma of olfactory receptor neurons (Nakamura and Gold, 1987;Firestein et al, 1991b). Presence of a low K,, dnc PDE in neuronal compartments outside of the cilia may be required for the modulation of this and other ion channels by CAMP.…”

Cyclic Nucleotide-Gated Ion Channels

“…In neurons of Helix, Aplysia, and Pleurobranchaea, the current persists in the presence of inhibitors of PKA (165,192,382), arguing that the cAMP action on channels is direct and does not involve phosphorylation. In excised membrane patches, perfusion with 1 mM cAMP produces single-channel events of ϳ40 pS (382). These two observations have been taken as evidence that the underlying channels belong to the class of CNG channels.…”

“…The unit conductance of 40 pS was determined in the presence of 60 mM divalent cations, and the maximum P o at saturating cAMP concentrations was only 6.3% (at 0 mV; Ref. 382). Known CNG channels, whether vertebrate or invertebrate, are strongly blocked by 60 mM divalents, and no single-channel recordings would be feasible.…”

Studies of the regulation and function of the Gsα gene Gnas using gene targeting technology

“…The amino terminus and switch 2 regions interact directly with β-subunit to form the heterotrimer (Lambright et al, 1996;Wall et al, 1995), while the carboxyl terminus is important for receptor interactions (Conklin et al, 1996;Grishina & Berlot, 2000;Mazzoni et al, 2000;Schwindinger et al, 1994;Simonds et al, 1989;Sullivan et al, 1987). cAMP, the major second messenger resulting from G s α signaling, mediates its effects through direct binding and stimulation of several molecules, including protein kinase A (PKA), cAMPregulated guanine nucleotide exchange factors (cAMP-GEFs) (de Rooij et al, 1998;Kawasaki et al, 1998), and ion channels (Sudlow et al, 1993;Wainger et al, 2001). PKA is a serine/ threonine kinase which activates physiological changes and gene expression through direct phosphorylation of enzymes involved in intermediary metabolism and transcription factors such as the cAMP response element binding protein (CREB) (Montminy, 1997).…”