Gabriela Gonzalez

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.

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.

Pharmaco-topology of Sulfonylurea Receptors SEPARATE DOMAINS OF THE REGULATORY SUBUNITS OFK ATP CHANNEL ISOFORMS ARE REQUIRED FOR SELECTIVE INTERACTION WITH K+ CHANNEL OPENERS

The differential responsiveness of (SUR1/KIR6.2)4 pancreatic β-cellversus (SUR2A/KIR6.2)4 sarcolemmal or (SUR2B/KIR6.0)4 smooth muscle cellK ATP channels to K+ channel openers (KCOs) is the basis for the selective prevention of hyperinsulinemia, myocardial infarction, and acute hypertension. KCO-stimulation ofK ATP channels is a unique example of functional coupling between a transport ATPase and a K+ inward rectifier. KCO binding to SUR is Mg-ATP-dependent and antagonizes the inhibition of (KIR6.0)4 pore opening by nucleotides. Patch-clamping of matched chimeric human SUR1-SUR2A/KIR6.2 channels was used to identify the SUR regions that specify the selective response of sarcolemmalversus β-cell channels to cromakalim or pinacidilversus diazoxide. The SUR2 segment containing the 12th through 17th predicted transmembrane domains, TMD12–17, confers sensitivity to the benzopyran, cromakalim, and the pyridine, pinacidil, whereas an SUR1 segment which includes TMD6–11 and the first nucleotide-binding fold, NBF1, controls responsiveness to the benzothiadiazine, diazoxide. These data are incorporated into a functional topology model for the regulatory SUR subunits ofK ATP channels.

Two Regions of Sulfonylurea Receptor Specify the Spontaneous Bursting and ATP Inhibition of KATP Channel Isoforms

KATP channels are heteromultimers of KIR6.2 and a sulfonylurea receptor, SUR, an ATP binding cassette (ABC) protein with several isoforms. KIR6.2 forms a channel pore whose spontaneous activity and ATP sensitivity are modulated by the receptor via an unknown interaction(s). Side by side comparison of single-channel kinetics and steady-state ATP inhibition of human β-cell, SUR1/KIR6.2,versus cardiac, SUR2A/KIR6.2 channels demonstrate that the latter have a greater mean burst duration and open probability in the absence of nucleotides and ∼4-fold higher IC50(ATP). We have used matched chimeras of SUR1 and SUR2A to show that the kinetics, which determine the maximal open probability (Pomax), and the ATP sensitivity are functionally separable and to identify the two segments of SUR responsible for these isoform differences. A region within the first five transmembrane domains specifies the interburst kinetics, whereas a C-terminal segment determines the sensitivity to inhibitory ATP. The separable effects of SUR on ATP inhibition and channel kinetics implies that the cytoplasmic C terminus of SUR either directly modulates the affinity of a weak ATP binding site on the inward rectifier or affects linkage between the binding site and the gate. This is the first identification of parts of an ABC protein that interact with an ion channel subunit to modulate the spontaneous activity and ATP sensitivity of the heteromeric channel.

The tolbutamide site of SUR1 and a mechanism for its functional coupling to KATP channel closure

Micromolar concentrations of tolbutamide will inhibit (SUR1/KIR6.2)4 channels in pancreatic β‐cells, but not (SUR2A/KIR6.2)4 channels in cardiomyocytes. Inhibition does not require Mg2+ or nucleotides and is enhanced by intracellular nucleotides. Using chimeras between SUR1 and SUR2A, we show that transmembrane domains 12–17 (TMD12‐17) are required for high‐affinity tolbutamide inhibition of KATP channels. Deletions demonstrate involvement of the cytoplasmic N‐terminus of KIR6.2 in coupling sulfonylurea‐binding with SUR1 to the stabilization of an interburst closed configuration of the channel. The increased efficacy of tolbutamide by nucleotides results from an impairment of their stimulatory action on SUR1 which unmasks their inhibitory effects. The mechanism of inhibition of β‐cell KATP channels by sulfonylureas during treatment of non‐insulin‐dependent diabetes mellitus thus involves two components, drug‐binding and conformational changes within SUR1 which are coupled to the pore subunit through its N‐terminus and the disruption of nucleotide‐dependent stimulatory effects of the regulatory subunit on the pore. These findings uncover a molecular basis for an inhibitory influence of SUR1, an ATP‐binding cassette (ABC) protein, on KIR6.2, a ion channel subunit.

Sulfonylurea receptors set the maximal open probability, ATP sensitivity and plasma membrane density of KATP channels

KATP channels are heteromultimers of SUR and KIR6.2. C‐terminal truncation of KIR6.2 allows surface expression of the pore. KIR6.2ΔC35 channels display ∼7‐fold lower maximal open probability, ∼35‐fold reduced ATP sensitivity, reduced mean open time, a markedly increased transition rate from a burst into a long‐lived closed state, and have no counterpart in vivo. SUR1 and SUR2A restore wild‐type bursting, ATP sensitivity and increase channel density in the plasma membrane. The high IC50(ATP) of ∼4 mM for KIR6.2ΔCK185Q channels results from the additive effects of SUR removal and KIR6.2 modification. The results demonstrate allosteric interaction(s) are essential for normal intrinsic activity, ATP inhibition, and trafficking of KATP channels.

Hetero-concatemeric KIR6.X4/SUR14 channels display distinct conductivities but uniform ATP inhibition.

KIR6.1 and KIR6.2 are the pore-forming subunits ofK NDP , the nucleotide-diphosphate-activated K ATP channels, and classical K ATP channels, respectively. “Hybrid” channels, in which the structure is predetermined by concatemerizing KIR6.1 and KIR6.2, exhibit distinct conductivities specified by subunit number and position. Inclusion of one KIR6.2 is sufficient to open KIR6.X-X-X-X/SUR14 in the absence of nucleotide stimulation through sulfonylurea receptor-1 (SUR1). ATP inhibited the spontaneous bursting of hybrid channels with an IC50(ATP) ∼10− 5 m, similar to that of KIR6.24-containing channels. These findings and a transient increase in K NDP channel activity following rapid wash-out of MgATP suggested that KIR6.1 is not ATP-insensitive as previously believed. We propose that SUR-dependent, inhibitory ATP-enhanced interactions of the cytoplasmic domains of both KIR6.1 and KIR6.2 stabilize a closed form of the M2 bundle in the gating apparatus.