Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular Mg²⁺] (Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996