Publisher Summary ATP-sensitive potassium channels, or KATP channels, couple changes in cellular metabolism with membrane electrical activity. In pancreatic β-cells these channels set the resting membrane potential. Increased glucose metabolism changes the ATP/ADP ratio reducing the opening of K ATP channels causing membrane depolarization and activation of voltage-gated Ca 2+ channels. The resulting increase in [Ca 2+ ] i stimulates insulin exocytosis. Pharmacologically distinct types of K ATP channels have been identified in muscle cells, where their opening would be expected to reduce electrical activity. KATP channels are assembled from sulfonylurea receptors, SURs, members of the ATP-binding cassette superfarnily, and members of the inwardly rectifying potassium channel family, K IR 6.x. Genetic, biochemical, and electrophysiologic data establish that the β-cell channel is assembled from SUR1 and K IR 6.2. K IR 6.2 forms the pore of the channel, while SUR1 regulates channel activity and confers responsiveness to channel openers like diazoxide, pinacidil, and cromakalim, and to channel blockers, sulfonylureas, like tolbutamide and glibenclamide. This chapter describes methods used in the characterization of K ATP channels, including synthesis and radioiodination of two derivatives of glibenclamide that photolabel SUR1.

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATP channels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATP channels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATP channels to traffic to the plasma membrane.

This chapter describes the different biochemical and molecular steps used to identify, purify, and clone the sulfonylurea receptor. It also discusses the methods for the expression and partial purification of a histidine tagged sulfonylurea receptor (SUR)1 and for the reconstitution of the β-cell ATP-sensitive K+ channel from SUR1 and KIR 6.2. Glucose-stimulated insulin secretion from pancreatic beta (β) cells depends on the closure of ATP-sensitive K+ channels (KAxp). These channels are regulated by changes in the ratio of [ADP] to [ATP] that result from glucose metabolism. They are pharmacologically regulated by sulfonylurea agents, used in the treatment of non-insulin-dependent diabetes (NIDDM) and diazoxide, used in the treatment of hypoglycemic states such as familial hyperinsulinism.

Adenosine 5'-triphosphate-sensitive potassium (KATP) channels couple metabolic events to membrane electrical activity in a variety of cell types. The cloning and reconstitution of the subunits of these channels demonstrate they are heteromultimers of inwardly rectifying potassium channel subunits (KIR6.x) and sulfonylurea receptors (SUR), members of the ATP-binding cassette (ABC) superfamily. Recent studies indicate that SUR and KIR6.x associate with 1:1 stoichiometry to assemble a large tetrameric channel, (SUR/KIR6.x)4. The KIR6.x subunits form the channel pore, whereas SUR is required for activation and regulation. Two KIR6.x genes and two SUR genes have been identified, and combinations of subunits give rise to KATP channel subtypes found in pancreatic beta-cells, neurons, and cardiac, skeletal, and smooth muscle. Mutations in both the SUR1 and KIR6.2 genes have been shown to cause familial hyperinsulinism, indicating the importance of the pancreatic beta-cell channel in the regulation of insulin secretion. The availability of cloned KATP channel genes opens the way for characterization of this family of ion channels and identification of additional genetic defects.

Persistent hyperinsulinemic hypoglycemia of infancy is caused by inappropriate and excessive secretion of insulin. Although the disease is rare in outbred communities (approximately 1 case per 50,000 persons), the incidence is approximately 1 per 2500 in inbred Arabic communities in which there is a familial (autosomal recessive) form of the disease. The disease most commonly presents with severe hypoglycemia a few hours after birth, although some cases present after several weeks or months. Some patients have a response to treatment with diazoxide or somatostatin, but others require partial pancreatectomy to control the hyperinsulinism.1–3 It has recently been suggested2, . . .

ATP-sensitive potassium channels (KATP channels) are heteromultimers of sulfonylurea receptors (SUR) and inwardly rectifying potassium channel subunits (KIR6.x) with a (SUR–KIR6.x)4 stoichiometry. Association is specific for KIR6.x and affects receptor glycosylation and cophotolabeling of KIR6.x by 125I-azidoglibenclamide. Association produces digitonin stable complexes with an estimated mass of 950 kDa. These complexes can be purified by lectin chromatography or by using Ni2+–agarose and a his-tagged SUR1. Expression of SUR1∼(KIR6.2)i fusion constructs shows that a 1:1 SUR1:KIR6.2 stoichiometry is both necessary and sufficient for assembly of active KATP channels. Coexpression of a mixture of strongly and weakly rectifying triple fusion proteins, rescued by SUR1, produced the three channel types expected of a tetrameric pore.

The high-affinity sulfonylurea receptor, a novel member of the ATP-binding cassette superfamily, is one component of the ATP-sensitive K+ channel. The protein is critical for regulation of insulin secretion from pancreatic beta-cells, and mutations in the receptor have been linked to familial hyperinsulinemia, a disorder characterized by unregulated insulin release despite severe hypoglycemia. The sulfonylurea receptor is present in membranes from a number of endocrine and neuroendocrine cell lines, including HIT-T15, RINm5f, alpha TC-6, AtT-20, and GH3 cells. Two forms of the receptor are present in RINm5f and alpha TC-6 cells, with apparent SDS gel molecular masses of 140 and 150 kDa. The two forms have equally high affinity, KD approximately 3 nM, for an iodinated derivative of glyburide, an anti-diabetic sulfonylurea. The receptor is a glycoprotein; treatment of RINm5f or alpha TC-6 cells with tunicamycin reduces the 140 and 150 kDa species to a single approximately 137 kDa protein. The 140 and 150 kDa receptors bind differentially to concanavalin A and wheat germ agglutinin, and lectin-affinity chromatography is ideal for the initial stages of receptor purification. After lectin-affinity chromatography, the same methods can be applied for purifying the 150 kDa form as for the 140 kDa receptor. A transiently expressed receptor with a histidine-tagged carboxy-terminus was purified by Ni-agarose chromatography, and this variant was used to demonstrate that the 140 kDa polypeptide is full length. Anti-peptide antibodies directed against the amino-terminus of the receptor and antibodies against the nucleotide binding folds immunoprecipitate both receptor forms. The results indicate the 140 and 150 kDa receptors are differentially glycosylated forms of the same polypeptide chain.

Familial hyperinsulinism (HI) is a disorder of pancreatic beta-cell function characterized by persistent hyperinsulinism despite severe hypoglycemia. To define the molecular genetic basis of HI in Ashkenazi Jews, 25 probands were screened for mutations in the sulfonylurea receptor (SUR1) gene by single-strand conformation polymorphism (SSCP) analysis of genomic DNA and subsequent nucleotide sequence analyses. Two common mutations were identified: (I) a novel in-frame deletion of three nucleotides (nt) in exon 34, resulting in deletion of the codon for F1388 (delta F1388) and (II) a previously described g-->a transition at position-9 of the 3' splice site of intron 32 (designated 3992-9g-->a). Together, these mutations are associated with 88% of the HI chromosomes of the patients studied. 86Rb+ efflux measurements of COSm6 cells co-expressing Kir6.2 and either wild-type or delta F1388 SUR1 revealed that the F1388 mutation abolished ATP-sensitive potassium channel (KATP) activity in intact cells. Extended haplotype analyses indicated that the delta F1388 mutation was associated with a single specific haplotype whereas the 3992-9g-->a mutation was primarily associated with a single haplotype but also occurred in the context of several other different haplotypes. These data suggest that HI in Ashkenazi Jews is predominantly associated with mutations in the SUR1 gene and provide evidence for the existence of at least two founder HI chromosomes in this population.

We have cloned an isoform of the sulfonylurea receptor (SUR), designated SUR2. Coexpression of SUR2 and the inward rectifier K+ channel subunit Kir6.2 in COS1 cells reconstitutes the properties of K-ATP channels described in cardiac and skeletal muscle. The SUR2/Kir6.2 channel is less sensitive than the SUR/Kir6.2 channel (the pancreatic beta cell K-ATP channel) to both ATP and the sulfonylurea glibenclamide and is activated by the cardiac K-ATP channel openers, cromakalim and pinacidil, but not by diazoxide. In addition, SUR2 binds glibenclamide with lower affinity. The present study shows that the ATP sensitivity and pharmacological properties of K-ATP channels are determined by a family of structurally related but functionally distinct sulfonylurea receptors.

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.