Figure 4.

Serine → alanine mutation at PKA phosphorylation sites reduces channel’s interaction with PIP₂. (A) Membrane topology of ROMK1 in relationship to PIP₂ in the inner leaflet of the membrane. Like all inward-rectifier K⁺ channels, it consists of a short N-terminal cytoplasmic domain, two transmembrane domains, one partial membrane domain, and a long C-terminal cytoplasmic tail. The proximal C-terminal region of channel (amino acid 180–223 of ROMK1) contains many conserved basic residues that form the putative PIP₂-binding region. A critical role for Arg-188 (R188) in forming an electrostatic interaction with PIP₂ is described in the text. Ser-219 (S219) and Ser-313 (S313) are PKA sites that are involved in enhancing PIP₂ affinity (see text for details). (B) Membrane patches were excised and stabilized in FVPP solution. Anti-PIP₂ antibodies (40 nM in FVPP) were applied to inhibit the channels. The on-cell currents for wild-type (WT) and mutant channels were normalized and superimposed. Time bar is 120 sec. (C) Half-time (t_1/2) for maximal inhibition by anti-PIP₂ antibody (40 nM) for the wild-type (WT) and the mutant channels of ROMK1. Mean ± SEM, n = 4–16 for each group. ∗ indicates P < 0.01 by unpaired t test, S219D vs. S219A. ∗∗ indicates statistically insignificant, S313D vs. S313A.