Modulation of ion channels by cannabinoid receptors in rat sympathetic neurons

Abstract

The rat brain-cannabinoid receptor (CBl) is a member of the G protein coupled receptor family. The present study direcdy- tested the functional coupling of CB 1 receptor with neuronal ion channels. _ The rat CBl receptor.was heterologously expressed by microinjection of cRNA into the enzymatically dissociated adult rat superior cervical ganglion (SCG) neurons. The cannabinoid receptor agonists WIN 55,212-2 and .CP 55940 produced a voltagedependent inhibitio_n of whole cell Ca2+ currents. The maximal inhibitions of the Ca2+ current by WIN 55,212-2 and CP 55,940 were 73% and 38%, respectively. The ECso -of WIN 55,212-2 was 47 nM and the ECso of CP 55940 was 7 nM. The inhibition was mediated by pertussis toxin (PTX) sensitive Gprotein and the N-type - Ca2+ channels are the targets. The L-type Ca2+ channels, M type K+ current and the A current were not modulated by WIN 55,212-2. An inwardly rectifying current was activated by WIN 55,212-2. The selective CBl receptor antagonist SR 141716A and LY 320135 abolished the inhibition of the Ca2+ currents by WIN 55,212-2. However, when given alone, SR 141716A and LY 320135 increased the Ca2+ current in SCG neurons expressing the- CBl receptor. SR 141716A increased Ca2+ current with an ECso of 32 nM and a maximal current increase of 41 % at 1 μM. This increase of Ca2+ current by SR 141716A was a reversal of a tonic inhibition of Ca2+ current in neurons expressing CB 1 receptors. A K 192A mutant of the human CB 1 receptor was tested to determine whether the tonic activation of the cannabinoid receptor is due to endogenous arachidonyl ethanolamide (anandamide). The K192A mutant receptor was expressed by microinjection of receptor cDNA into nucleus of the SCG neurons. WIN 55,212-2 inhibited the Ca2+ current in these SCG neurons, but SR 141716A did not increase the Ca2+ current. However, SR 141716A inhibited the effect of WIN 55,212-2. Ca2+ ·currents from male rat major pelvic ganglion neurons were examined for modulation by native cannabinoid receptors. WIN 55,212-2 inhibited and SR· 141716A increased the voltage-dependent Ca2+ currents in a subpopulation of the rat major pelvic ganglion neurons. 1 μM WIN 55,212-2 inhibited current by 26.1±1.8% and 1 μM SR 141716A increased current by 27.4±6.9%. These findings indicate that both heterologously expressed CB 1 cannabinoid receptors and the native neuronal cannabinoid receptors can inhibit voltage-dependent - Ca2+ channels. There is a tonic activation of both the heterologously expressed rat and human CB 1 receptor and the native rat cannabinoid receptor. Two possible mechanisms may be responsible for the tonic receptor activation: an endogenous agonist may exist or the cannabinoid receptor can adopt an active conformational state such that SR 141716A may act as an inverse agonist.