Na⁺-K⁺-ATPase-G protein coupling in myocardial sarcolemma: separation and reconstitution

¹ Laboratory of Molecular Endocrinology, USSR Cardiology Research Center, Moscow 121552, USSR

The cholinergic agonist carbachol produces a concentration-dependent (half-maximum inhibitory concentration = 0.9 µM) decrease in the Na⁺-K⁺-adenosine triphosphatase (ATPase) activity of rabbit cardiac sarcolemma that occurred only in the presence of guanosine 5'-[-thio]triphosphate (0.1 µM GTPS) and reached 40% inhibition. The inhibition is blocked by the muscarinic receptor antagonist atropine (10 µM) and is abolished in sarcolemma treated with pertussis toxin (20 µg/ml) in the presence of 100 µM NAD. GTPS alone reduces Na⁺-K⁺-ATPase activity by 45% (half-maximum inhibitory = 1 µM). The apparent affinity of the enzyme for GTPS is increased 10-fold in the presence of 1 µM carbachol. In sarcolemma solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS, 10 mM), the GTPS-dependent inhibition of the Na⁺-K⁺-ATPase is also observed. Gel filtration of a CHAPS extract of sarcolemma on a Sepharose CL-6B column resulted in a separation of Na⁺-K⁺-ATPase and pertussis toxin-sensitive G_i activities. Na⁺-K⁺-ATPase activity that was separated on the column lost its sensitivity to the inhibitory action of guanine nucleotides. Inhibitory effects (20–30%) of guanosine 5'-triphosphate analogues [Gpp(NH)p, GTPS, or Gpp(CH₂)p] at micromolar concentrations were restored when the Na⁺-K⁺-ATPase activity was recombined with fractions that contained the pertussis toxin-sensitive G_i protein(s). Similar concentrations of guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine-5'-[-thio]diphosphate, or App(NH)p were unable to induce the G_i protein-mediated attenuation of Na⁺-K⁺-ATPase activity in the reconstitution system. These results suggest that a pertussis toxin-sensitive G_i protein may act as a transducer of the inhibitory hormonal signals on Na⁺-K⁺-ATPase in the sarcolemma.