1,4‐Dihydropyridines modulate GTP hydrolysis by G_o in neuronal membranes

Abstract

Several lines of evidence suggest that L-type Ca :~ channels (I ,4-dihydropyridin¢ receptors) are modulated by GTP-binding proteins. We have further examined this interaction by measuring the effect of 1,4-dihydropyridlnes on GTPase activity in brain membranes. Dihydropyridine agonists significantly increased GTPase, reflected by an increase in the maximal rate of GTP hydrolysis, without affecting the affinity for GTP or the binding ofa non-hydrolysable analogue of GTP.

Inhibition of the interaction of G protein G(o) with calcium channels by the calcium channel beta‐subunit in rat neurones.

“…(-)-Baclofen (50 #M) inhibited 18-9 + 6-7 % (n = 8) of the residual current following irreversible inhibition by w-CgTX GVIA, indicating that L-type IBa was also inhibited by this agonist, although to a smaller extent than N-type IBa. The proportion of IBa that was w-CgTX GVIA sensitive was not affected by oligonucleotide injection ( (Sweeney & Dolphin, 1992;1995). We interpreted this effect as indicating that L-type VDCCs in the presence of a DHP agonist stimulate the intrinsic GTPase activity of G0.…”

“…We interpreted this effect as indicating that L-type VDCCs in the presence of a DHP agonist stimulate the intrinsic GTPase activity of G0. We put forward the hypothesis that VDCCs may be acting as GTPase-activating proteins (GAPs) for G. (Sweeney & Dolphin, 1992). In order to determine whether the VDCC fl-subunit is involved in this process, we have now extended these studies by investigating the effect of our antipeptide antibody against the VDCC fl-subunit on low Km GTPase activity in rat brain membranes stimulated by the calcium channel agonist isomer S-(-)-Bay K 8644.…”

“…(-)-Baclofen was obtained from Ciba-Geigy (Bazel, Switzerland), o-conotoxin (w-CgTX) GVIA from Peninsula Labs (Belmont, CA, USA), and nicardipine from Syntex (Palo Alto, CA, USA). GTPase Preparation of rat frontal cortical membranes and GTPase assay were performed exactly as described previously (Sweeney & Dolphin, 1992), except that the S-(-)-isomer of Bay K 8644 (RBI Biochemicals, USA) was used rather than the racemate. Membranes were preincubated with antiserum, or preimmune serum, for 1 h at 30°C at a 1:50 dilution.…”

Voltage‐dependent calcium channel β‐subunits in combination with α1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by Gαo in rat frontal cortex

“…The frontal cortex membrane preparation and GTPase assay were performed as described previously [22].…”

“…However, GTPase can also be potentiated by an effector protein acting as a GTPase-activating-protein (GAP) to stimulate the intrinsic GTPase activity [19,20,21]. It has been shown previously that the GTPase activity of the G-protein Go can be stimulated by a number of DHP agonists [22]. Using an antipeptide anti-VDCC fl-subunit antiserum and a peptide, which mimics the fl subunit binding site on the VDCC c~-subunit [23], we now provide evidence that the VDCC fl-subunit is the principal component of the L-type calcium channel involved in linking DHP agonist binding with enhanced GTPase activity of the L-type VDCC associated G-protein.…”