letters to nature and 10 Na3HP2O7. FV solution also contained 0.2 NaF and 0.1 Na3VO4. Rarely, irreversible current rundown still occurred with FVPP. The total Na+ concentration of all cytoplasmic solutions was adjusted to 30 mM with NaOH, and pH was adjusted to 7.0 with N-methylglucamine (NMG) or HCl. PIP2 liposomes (20–200 nm) were prepared by sonicating 1 mM PIP2 (Boehringer Mannheim) in distilled water. Reconstituted monoclonal PIP2 antibody (Perspective Biosystems, Framingham, MA) was diluted 40-fold into experimental solution. Current–voltage relations of all currents reversed at EK and showed characteristic rectification, mostly owing to the presence of Na+ in FVPP and possibly also residual polyamines. Current records presented (measured at 30 C, −30 mV holding potential) are digitized strip-chart recordings. Purified bovine brain Gbg29 was diluted just before application such that the final detergent (CHAPS) concentration was 5 M. Detergent-containing solution was washed away thoroughly before application of PIP2, because application of phospholipid vesicles in the presence of detergent usually reversed the effects of Gbg; presumably, Gbg can be extracted from membranes by detergent plus phospholipids. Molecular biology. R188Q mutation was constructed by insertion of the mutant oligonucleotides between the Bsm1 and BglII sites of pSPORT– ROMK1 (ref. 11). A polymerase chain reaction (PCR) fragment (amino acids 180–391) from pSPORT–ROMK1 R188Q mutant was subcloned into pGEX2T vector (Pharmacia) for expression of R188Q mutant protein of GST–RKC. The construction, expression and purification of GST–IKC (amino acids 182– 428 of IRK1), GST–GKC (180–462 of GIRK1), GST–IKN (1–86 of IRK1) have been described21,22. 3 In vitro PIP2 binding assay. H-PIP2 in chloroform-methanol (1:1) (American Radiolabeled Chemicals; 0.4 Ci nM−1 specific activity) was dried under N2 and