NEUROSTEROIDS MODULATE CALCIUM CURRENTS IN HIPPOCAMPAL CA1 NEURONS VIA A PERTUSSIS-TOXIN-SENSITIVE G-PROTEIN-COUPLED MECHANISM

The inhibition of Ca2+ channel currents by endogenous brain steroids w as examined in freshly dissociated pyramidal neurons from the adult gu inea pig hippocampal CA1 region. The steady-state inhibition of the pe ak Ca2+ channel current evoked by depolarizing steps from -80 to -10 m V occurred in a concentration-dependent manner with the following IC50 values: pregnenolone sulfate (PES), 11 nM; pregnenolone (PE), 130 nM; and allotetrahydrocorticosterone (THCC), 298 nM. THCC, PE, and PES de pressed a fraction of the Ca2+ channel current with a maximal inhibiti on of 60% of the total current. However, substitution of an acetate gr oup for the sulfate group on PES resulted in a complete loss of activi ty. Progesterone had no effect (4% inhibition at 100 mu M) Intracellul ar dialysis of PES had no effect on the Ca2+ current; concomitant extr acellular perfusion of PES showed normal inhibitory activity, suggesti ng that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES sl owed the rate of Ca2+ current activation and deactivation with no chan ge in the voltage dependence of activation. Inhibition of the Ca2+ cha nnel current by THCC and PES was voltage dependent. THCC primarily inh ibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel curr ent. PE was nonselective in inhibiting both the CgTX- and the nifedipi ne (NIF)-sensitive Ca2+ channel current. These neurosteroids had no ef fect on the CgTX/NIF-insensitive current. In neurons isolated from per tussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibitio n by PES, PE, and THCC was significantly diminished. Intracellular dia lysis with GDP-beta-S (500 mu M) also significantly diminished the neu rosteroid inhibition of the Ca2+ channel current. Intracellular dialys is with the general kinase inhibitors H-7 (100 mu M), staurosporine (4 00 nM), and a 20 amino acid protein kinase inhibitor(1 mu M) also sign ificantly prevented the THCC and PES inhibition of the Ca2+ channel cu rrent. Intracellular dialysis with the more specific inhibitors of pro tein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19-36) (1-2 m u M) and bisindolylmaleimide (1 mu M) significantly diminished the THC C and PE inhibition of the Ca2+ channel current. Rp-cAMPS (100 mu M), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no ef fect on the THCC and PE inhibition of the Ca2+ current. These results demonstrate that neurosteroids, acting at a membrane receptor site, ar e potent modulators of either the CgTX- and/or the NIF-sensitive Ca2channel current but not the CgTX/NIF-insensitive current. Furthermore, the Ca2+ channel current inhibition is via a PTX-sensitive G-protein- coupled mechanism associated with the activation of PKC. Thus, inhibit ion of Ca2+ channel currents by neurosteroids may participate in the r egulation of synaptic processes such as modulation of neuronal activit y and/or neurotransmitter release-associated mechanisms via a G-protei n mechanism(s).