Na⁺ promotes the dissociation between Gα_GDP and Gβγ, activating G protein-gated K⁺ channels

G protein-gated K⁺ channels (GIRK, or Kir3) are activated by the direct binding of Gβγ or of cytosolic Na⁺.Na⁺ activation is fast, Gβγ-independent, and probably via a direct, low affinity (EC₅₀, 30-40 mM) binding of Na⁺ to the channel. Here we demonstrate that an increase in intracellular Na⁺ concentration, [Na⁺]_in, within the physiological range (5-20 mM), activates GIRK within minutes via an additional, slow mechanism. The slow activation is observed in GIRK mutants lacking the direct Na⁺ effect. It is inhibited by a Gβγ scavenger, hence it is Gβγ-dependent; but it does not require GTP. We hypothesized that Na⁺ elevates the cellular concentration of free Gβγ by promoting the dissociation of the Gαβ heterotrimer into free Gα_GDP and Gβγ. Direct biochemical measurements showed that Na⁺ causes a moderate decrease (∼2-fold) in the affinity of interaction between Gα_GDP and Gβγ. Furthermore, in accord with the predictions of our model, slow Na⁺ activation was enhanced by mild coexpression of Gα_i3. Our findings reveal a previously unknown mechanism of regulation of G proteins and demonstrate a novel Gβγ-dependent regulation of GIRK by Na⁺. We propose that Na⁺ may act as a regulatory factor, or even a second messenger, that regulates effectors via Gβγ.