Molecular mechanism for sodium-dependent activation of G protein-gated K+ channels

Abstract

1. G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gbetagamma and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1-GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1-GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4, 5-bisphosphate (PIP2). 2. The rate of inhibition of GIRK1-GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. 3. The dose-response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. 4. Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. 5. We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2.

Figure 1. The effect of negative charges at position 226/217 on the basal activity of the wild-type GIRK1–GIRK2 (G1/G2) and the mutant GIRK1–GIRK2D226N (4N) and GIRK1N217D-GIRK2 (4D) heteromeric channels

A, continuous currents at −80 mV, recorded by two-electrode voltage clamp from Xenopus oocytes expressing the indicated channels and the m₂ muscarinic receptor. Solution changes are indicated by the bars above the current traces. CCh (carbachol), 3 μM; Ba²⁺, 1 mM. Dotted line indicates zero current level. B, Ba²⁺-sensitive basal and 3 μM CCh-induced current amplitudes at −80 mV (n = 6 for each group). C, basal currents expressed as a fraction of the total current in the presence of 3 μM CCh minus the Ba²⁺-insensitive current, for the wild-type and mutant channels. * Values significantly different from each other, P < 0.001 (n = 6 for each group).

Figure 2. Inhibitory effect of anti-PIP₂ antibody on the wild-type GIRK1–GIRK2 and the mutant GIRK1–GIRK2D226N and GIRK1N217D-GIRK2 heteromeric channels

A, the anti-PIP₂ antibody was applied to inside-out patches in the continuous presence of 5 mM MgATP in FV solution with or without 60 mM Na⁺ as indicated. The time course of inhibition was fitted with single exponential curves. B, currents were normalized to the mean amplitude prior to antibody application, and overlayed. For clarity, the fitted curves for both GIRK1–GIRK2D226N and GIRK1N217D-GIRK2 channels are plotted, and the wild-type GIRK1–GIRK2 currents were smoothed. C, mean values of the time constants for the monoexponential fits. *P < 0.05 (n = 3–4 for each group).

Figure 3. dose–response relationship for activation of GIRK1–GIRK2 channels by purified PIP₂ liposomes in the absence and presence of internal Na⁺

A, GIRK1–GIRK2 channel activity ran down following patch excision into MgATP-free bath solution. C/A is cell-attached and I/O is inside-out configuration. Bars indicate the periods of application of the different reagents. Purified PIP₂ was applied for 3 min each time and the mean current was measured for the final minute of this period. B, dose–response relationship for PIP₂ in the absence and presence of Na⁺ and Mg²⁺. Currents were normalized to the cell-attached level. Asterisks indicate a significant difference from the current in the absence of PIP₂, Na⁺ and Mg²⁺ (*P < 0.01 and **P < 0.001; n = 3–15 for each group).

Figure 4. Activation of GIRK1–GIRK2 channels by synthetic PIP₂ analogues

The experimental protocol was the same as that shown in Fig. 3A. Currents were normalized to the cell-attached level. Asterisks indicate a significant difference from currents in the absence of PIP₂, Na⁺ and Mg²⁺ (*P < 0.05, **P < 0.01 and ***P < 0.001; n = 3–5 for each group).