Jmh. Ffrenchmullen et al., NEUROSTEROIDS MODULATE CALCIUM CURRENTS IN HIPPOCAMPAL CA1 NEURONS VIA A PERTUSSIS-TOXIN-SENSITIVE G-PROTEIN-COUPLED MECHANISM, The Journal of neuroscience, 14(4), 1994, pp. 1963-1977
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).