John P. Clement

Reconstitution of IKATP: An Inward Rectifier Subunit Plus the Sulfonylurea Receptor

A member of the inwardly rectifying potassium channel family was cloned here. The channel, called BIR (Kir6.2), was expressed in large amounts in rat pancreatic islets and glucose-responsive insulin-secreting cell lines. Coexpression with the sulfonylurea receptor SUR reconstituted an inwardly rectifying potassium conductance of 76 picosiemens that was sensitive to adenosine triphosphate (ATP) (IKATP) and was inhibited by sulfonylureas and activated by diazoxide. The data indicate that these pancreatic β cell potassium channels are a complex composed of at least two subunits-BIR, a member of the inward rectifier potassium channel family, and SUR, a member of the ATP-binding cassette superfamily. Gene mapping data show that these two potassium channel subunit genes are clustered on human chromosome 11 at position 11p15.1.

A FAMILY OF SULFONYLUREA RECEPTORS DETERMINES THE PHARMACOLOGICAL PROPERTIES OF ATP-SENSITIVE K+ CHANNELS

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 KATP 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.

Association and Stoichiometry of KATP Channel Subunits

ATP-sensitive potassium channels (K(ATP) channels) are heteromultimers of sulfonylurea receptors (SUR) and inwardly rectifying potassium channel subunits (K(IR)6.x) with a (SUR-K(IR)6.x)4 stoichiometry. Association is specific for K(IR)6.x and affects receptor glycosylation and cophotolabeling of K(IR)6.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 approximately (K(IR)6.2)i fusion constructs shows that a 1:1 SUR1:K(IR)6.2 stoichiometry is both necessary and sufficient for assembly of active K(ATP) 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.

Adenosine diphosphate as an intracellular regulator of insulin secretion

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.

The C Terminus of SUR1 Is Required for Trafficking of KATP Channels

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 KATPchannels 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 KATPchannels 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 KATPchannels to traffic to the plasma membrane.

SULFONYLUREA RECEPTORS AND ATP-SENSITIVE POTASSIUM ION CHANNELS

Publisher Summary 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 K IR 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.

ATP-SENSITIVE POTASSIUM CHANNELS

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.