Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple the cellular metabolic state to electrical activity and are a critical link between blood glucose concentration and pancreatic insulin secretion. A mutation in the second nucleotide-binding fold (NBF2) of the sulfonylurea receptor (SUR) of an individual diagnosed with persistent hyperinsulinemic hypoglycemia of infancy generated KATP channels that could be opened by diazoxide but not in response to metabolic inhibition. The hamster SUR, containing the analogous mutation, had normal ATP sensitivity, but unlike wild-type channels, inhibition by ATP was not antagonized by adenosine diphosphate (ADP). Additional mutations in NBF2 resulted in the same phenotype, whereas an equivalent mutation in NBF1 showed normal sensitivity to MgADP. Thus, by binding to SUR NBF2 and antagonizing ATP inhibition of KATP++ channels, intracellular MgADP may regulate insulin secretion.

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates KATP and other inward rectifier (Kir) channels. To determine residues important for PIP2 regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using 86Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176–222 and 301–314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP2. Only one residue (R201A) simultaneously affected ATP and PIP2 sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315–390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP2 sensitivity of KATP channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP2 sensitivity in other inward rectifier channels, indicating a common structural basis for PIP2 regulation.

The cellular prion protein (PrPC) is a glycolipid-anchored protein that is involved in the pathogenesis of fatal spongiform encephalopathies. We have shown previously that, in contrast to several other glycolipid-anchored proteins, chPrP, the chicken homologue of mammalian PrPC, is endocytosed via clathrin-coated pits in cultured neuroblastoma cells, as well as in embryonic neurons and glia (Shyng, S.-L., Heuser, J. E., and Harris, D. A. (1994) J. Cell Biol. 125, 1239-1250). In this study, we have determined that the N-terminal half of the chPrP polypeptide chain is essential for its endocytosis. Deletions within this region reduce the amount of chPrP internalized, as measured by surface iodination or biotinylation, and decrease its concentration in clathrin-coated pits, as determined by quantitative electron microscopic immunogold labeling. Mouse PrP, as well as two mouse PrP/chPrP chimeras, are internalized as efficiently as chPrP, suggesting that conserved features of secondary and tertiary structure are involved in interaction with the endocytic machinery. Our results indicate that the ectodomain of a protein can contain endocytic targeting information, and they strongly support a model in which the polypeptide chain of PrPC binds to the extracellular domain of a transmembrane protein that contains a coated pit localization signal in its cytoplasmic tail.

ATP-sensitive potassium (K(ATP)) channels are inhibited by intracellular ATP and activated by ADP. Nutrient oxidation in beta-cells leads to a rise in [ATP]-to-[ADP] ratios, which in turn leads to reduced K(ATP) channel activity, depolarization, voltage-dependent Ca(2+) channel activation, Ca(2+) entry, and exocytosis. Persistent hyperinsulinemic hypoglycemia of infancy (HI) is a genetic disorder characterized by dysregulated insulin secretion and, although rare, causes severe mental retardation and epilepsy if left untreated. The last five or six years have seen rapid advance in understanding the molecular basis of K(ATP) channel activity and the molecular genetics of HI. In the majority of cases for which a genotype has been uncovered, causal HI mutations are found in one or the other of the two genes, SUR1 and Kir6.2, that encode the K(ATP) channel. This article will review studies that have defined the link between channel activity and defective insulin release and will consider implications for future understanding of the mechanisms of control of insulin secretion in normal and diseased states.

ATP-sensitive potassium channels (KATP) are unique among K¤ channels in being rapidly and reversibly inhibited by the non-hydrolytic binding of cytoplasmic adenine nucleotides (Noma, 1983;Ashcroft, 1988;. The realization that these channels are encoded by an ATP binding cassette (ABC) protein, the sulphonylurea receptor (SUR1 or SUR2), in addition to an inward rectifier K¤ channel subunit (Kir6.1 or Kir6.2; Inagaki et al. 1995Inagaki et al. , 1996 led to the natural presumption that inhibitory ATP binding should occur at the nucleotide binding folds (NBFs) of the SUR subunit. Mutagenic studies have now demonstrated that the NBFs of SUR1 control activation of the channel by Mg¤-bound nucleotides and diazoxide, probably as a consequence of these agents mimicking, or enhancing, an effect of nucleotide hydrolysis (Nichols et al. 1996;Gribble et al. 1997;Shyng et al. 1997b). However, none of these studies has demonstrated an effect of SUR1 mutations on the intrinsic sensitivity of expressed channels to ATP inhibition. On the other hand, tandem dimeric constructs of SUR1 and Kir6.2 express channels with reduced ATP sensitivity (Clement et al. 1997;Shyng & Nichols, 1997), and, moreover, mutations of Kir6.2 can alter the inhibitory effect of ATP on channels formed by co-expression with SUR1 (Shyng et al. 1997a;Tucker et al. 1997Tucker et al. , 1998. In the study of Shyng et al. (1997a), mutations of asparagine 160 in the second transmembrane segment alter sensitivity to polyamine-induced rectification, as well as decreasing the apparent ATP sensitivity approximately 5-fold. Tucker et al. (1997) reported that a C-terminally truncated Kir6.2 subunit (Kir6.2ÄC36) could express ATPsensitive channels in the absence of SUR1, and reported a significant reduction of ATP sensitivity for Kir6.2ÄC36 channels with an additional mutation of lysine 185 in the truncated C-terminus. Lorenz et al. (1998) 1. To gain insight into the role of the cytoplasmic regions of the Kir6.2 subunit in regulating channel activity, we have expressed the sulphonylurea receptor SUR1 with Kir6.2 subunits containing systematic truncations of the N-and C-termini. Up to 30 amino acids could be truncated from the N-terminus, and up to 36 amino acids from the C-terminus without loss of functional channels in co-expression with SUR1. Furthermore, Kir6.2ÄC25 and Kir6.2ÄC36 subunits expressed functional channels in the absence of SUR1. 2. In co-expression with SUR1, N-terminal truncations increased Ki,ATP ([ATP] causing halfmaximal inhibition of channel activity) by as much as 10-fold, accompanied by an increase in the ATP-insensitive open probability, whereas the C-terminal truncations did not affect the ATP sensitivity of co-expressed channels. 3. A mutation in the near C-terminal region, K185Q, reduced ATP sensitivity of co-expressed channels by approximately 30-fold, and on the Kir6.2ÄN2-30 background, this mutation decreased ATP sensitivity of co-expressed channels by approximately 400-fold. 4. Each of these mutations also reduced the sensitivity to inhibit...