Insulin secretagogues, sulfonylurea receptors and K(ATP) channels.

ATP-sensitive K+ channels, termed K(ATP) channels, provide a link between cellular metabolism and membrane electrical activity in a variety of tissues. Channel isoforms have been identified and are targets for compounds that both stimulate and inhibit their activity resulting in membrane hyperpolarization and depolarization, respectively. Examples include relaxation of vascular smooth muscle and stimulation of insulin secretion. This article reviews the cloning, molecular biology, and structure of K(ATP) channels, with particular focus on the SUR1/K(IR)6.2 neuroendocrine channels that are important for the regulation of insulin secretion. We integrate the extensive pharmacologic structure-activity-relationship data on these channels, which defines a bipartite drug binding pocket in the SUR (sulfonylurea receptor), with recent structure-function studies that identify domains of SUR and K(IR)6.2, the channel pore, which are critical for channel assembly, for gating, and for the ligand-receptor interactions that modulate channel activity. The atomic structure of a sulfonylurea in a protein pocket is used to develop insight into the recognition of these compounds. A homology model of K(ATP) channels, based on VC-MsbA, another member of the ABC protein family, is described and used to position amino acids important for the action of channel openers and blockers within the core of SUR. The model has a central chamber which could serve as a multifaceted binding pocket.

A conserved inhibitory and differential stimulatory action of nucleotides on K(IR)6.0/SUR complexes is essential for excitation-metabolism coupling by K(ATP) channels.

The mechanism by which ubiquitous adenine nucleotide-gated K(IR)6.0(4)/SUR(4) channels link membrane excitability with cellular metabolism is controversial. Is a decreased sensitivity to inhibitory ATP required, or is the Mg-ADP/ATP-dependent stimulatory action of the ATPase, sulfonylurea receptor (SUR), on K(IR) sufficient to elicit a physiologically significant open channel probability? To evaluate the roles of nucleotide inhibition versus stimulation, we compared K(IR)6.1-based K(NDP) channels with K(IR)6.2-based K(ATP) channels and all possible K(IR)6.1/6.2 hybrids. Although K(NDP) channels are thought to be poorly sensitive to inhibitory ATP and to require Mg-nucleotide diphosphates for activity, we demonstrate that, like K(ATP), and hybrid channels, they are inhibited with an IC(50(ATP)) 100-fold lower than [ATP](i). K(IR)6.1 is, however, more efficiently stimulated by SUR than K(IR)6.2, thus providing a mechanism for differential nucleotide regulation, in addition to the known differential interactions of Mg-nucleotides with SUR isoforms. The on-cell and spontaneous activities of K(NDP), K(ATP), and hybrid channels identified in native cells, are different; thus, their similar IC(50(ATP)) values argue the regulatory "beta" SUR subunits play a preeminent role in coupling excitation to metabolism and pose questions about the physiologic significance of models, which assume the ATP insensitivity of open K(IR)s.

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

K(IR)6.1 and K(IR)6.2 are the pore-forming subunits of K(NDP)(,) the nucleotide-diphosphate-activated K(ATP) channels, and classical K(ATP) channels, respectively. "Hybrid" channels, in which the structure is predetermined by concatemerizing K(IR)6.1 and K(IR)6.2, exhibit distinct conductivities specified by subunit number and position. Inclusion of one K(IR)6.2 is sufficient to open K(IR)6. X-X-X-X/SUR1(4) in the absence of nucleotide stimulation through sulfonylurea receptor-1 (SUR1). ATP inhibited the spontaneous bursting of hybrid channels with an IC(50(ATP)) approximately 10(-)(5) m, similar to that of K(IR)6.2(4)-containing channels. These findings and a transient increase in K(NDP) channel activity following rapid wash-out of MgATP suggested that K(IR)6.1 is not ATP-insensitive as previously believed. We propose that SUR-dependent, inhibitory ATP-enhanced interactions of the cytoplasmic domains of both K(IR)6.1 and K(IR)6.2 stabilize a closed form of the M2 bundle in the gating apparatus.

Pharmaco-topology of sulfonylurea receptors. Separate domains of the regulatory subunits of K(ATP) channel isoforms are required for selective interaction with K(+) channel openers.

The differential responsiveness of (SUR1/K(IR)6.2)(4) pancreatic beta-cell versus (SUR2A/K(IR)6.2)(4) sarcolemmal or (SUR2B/K(IR)6. 0)(4) smooth muscle cell K(ATP) channels to K(+) channel openers (KCOs) is the basis for the selective prevention of hyperinsulinemia, myocardial infarction, and acute hypertension. KCO-stimulation of K(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 (K(IR)6.0)(4) pore opening by nucleotides. Patch-clamping of matched chimeric human SUR1-SUR2A/K(IR)6.2 channels was used to identify the SUR regions that specify the selective response of sarcolemmal versus beta-cell channels to cromakalim or pinacidil versus 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 of K(ATP) channels.

The tolbutamide site of SUR1 and a mechanism for its functional coupling to K(ATP) channel closure.

Micromolar concentrations of tolbutamide will inhibit (SUR1/K(IR)6. 2)(4) channels in pancreatic beta-cells, but not (SUR2A/K(IR)6.2)(4) channels in cardiomyocytes. Inhibition does not require Mg(2+) 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 K(ATP) channels. Deletions demonstrate involvement of the cytoplasmic N-terminus of K(IR)6.2 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 beta-cell K(ATP) 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 K(IR)6.2, a ion channel subunit.

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 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.

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 beta-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 approximately 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.

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. KIR6.2deltaC35 channels display approximately 7-fold lower maximal open probability, approximately 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 approximately 4 mM for KIR6.2deltaCK185Q 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.

The N-terminus of KIR6.2 limits spontaneous bursting and modulates the ATP-inhibition of KATP channels.

KATP channels are heteromultimers of a sulfonylurea receptor SUR and KIR6.2 with the inward rectifier forming the pore which is regulated by SUR. We have examined the contributions of the cytoplasmic domains of KIR6.2 to control of spontaneous bursting and ATP-inhibition in human SUR1/KIR6.2 KATP channels. Truncations of the N-terminus of KIR6.2 nearly eliminate transitions to interburst closed states without affecting the open or intraburst closed states, thus producing SUR1/DeltaNKIR6.2 channels with an extremely high open probability in the absence of nucleotides. These channels have a decrease apparent ATP-sensitivity which is consistent with the involvement of the N-terminus in a transition to an interburst closed state that preferentially binds inhibitory ATP. Mutations in both the N- and proximal C-termini of KIR6.2 can synergistically attenuate the ATP-inhibition. The results identify the N-terminus of KIR6.2 as a determinant of the interburst kinetics of KATP channels and suggest that the two cytoplasmic domains of KIR6.2 participate in ATP-inhibitory gating through distinct mechanisms.

Reconstituted human cardiac KATP channels: functional identity with the native channels from the sarcolemma of human ventricular cells.

ATP-sensitive potassium (KATP) channels in striated myocytes are heteromultimers of KIR6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human KIR6.2 (huKIR6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A+ mRNA. ATP- and glibenclamide-sensitive K+ channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous KATP channels, but not when huSUR2A or huKIR6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal KATP channels in human ventricular cells. The single-channel conductance of approximately 80 pS measured at -40 mV in quasi-symmetrical approximately 150 mmol/L K+ solutions, the intraburst kinetics that were dependent on K+ driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huKIR6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg2+ ( approximately 0. 7 mmol/L) or in the absence of Mg2+, with an apparent IC50 of approximately 20 micromol/L and a pseudo-Hill coefficient of approximately 1. They were "refreshed" by MgATP and stimulated by ADP in the presence of Mg2+ when inhibited by ATP. The huSUR2A/huKIR6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg2+ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huKIR6.2 channels represent KATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.

A view of sur/KIR6.X, KATP channels.

ATP-sensitive potassium channels, termed KATP channels, link the electrical activity of cell membranes to cellular metabolism. These channels are heteromultimers of sulfonylurea receptor (SUR) and KIR6.X subunits associated with a 1:1 stoichiometry as a tetramer (SUR/KIR6.X forms the pores, whereas SUR regulates their activity. Changes in [ATP]i and [ADP]i gate the channel. The diversity of KATP channels results from the assembly of SUR and KIR6.X subtypes KIR6.1-based channels differ from KIR6.2 channels mainly by their smaller unitary conductance. SUR1- and SUR2-based channels are distinguished by their differential sensitivity to sulfonylureas, whereas SUR2A-based channels are distinguished from SUR2B channels by their differential sensitivity to diazoxide. Mutations that result in the loss of KATP channels in pancreatic beta-cells have been identified in SUR1 and KIR6.2. These mutations lead to familial hyperinsulinism. Understanding the mutations in SUR and KIR6.X is allowing insight into how these channels respond to nucleotides, sulfonylureas, and potassium channel openers, KCOs.

Purinergic facilitation of ATP-sensitive potassium current in rat ventricular myocytes.

1. The effects of different purinergic agonists on the cardiac adenosine 5'-triphosphate (ATP)-sensitive potassium current (IK(ATP)), appearing during dialysis of rat isolated ventricular myocytes with a low-ATP (100 microM) internal solution under whole-cell patch-clamp conditions, were examined in the presence of a P1 purinoceptor antagonist. 2. The extracellular application of ATP in the micromolar range induced, besides known inward currents through cationic and chloride channels, the facilitation of IK(ATP) once IK(ATP) had already been partially activated during the low-ATP dialysis. 3. Analogues of ATP, alpha, beta-methyleneadenosine 5'-triphosphate (alpha, beta meATP), 2-methylthioadenosine triphosphate (2MeSATP), adenosine 5'-O-3-thiotriphosphate (ATP gamma S) similarly facilitated IK(ATP). UTP and ADP were very weak agonists while AMP and adenosine had no detectable effect. 4. The half-maximal stimulating concentration (C50) of alpha, beta meATP, an analogue that did not elicite the interfering inward cationic current was 1.5 microM. Similar apparent C50 (1-2 microM) were observed for ATP and analogues tested with somewhat less maximal effect of ATP gamma S. 5. Suramin, a nonselective P2-purinoceptor antagonist, altered IK(ATP) at the relatively high concentration required to inhibit purinoceptors. Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), a supposedly predominantly P2x-purinoceptor antagonist, at micromolar concentration inhibited the transient inward current but did not block the facilitation of IK(ATP). 6. Our results demonstrate that ATP and its analogues facilitate IK(ATP) in rat ventricular myocytes by stimulation of non-P1-, non-P2x-purinoceptors.

ATP-sensitive K(+)-channels in the human adult ventricular cardiomyocytes membrane.

Using the patch-clamp method in 27 different inside-out patches it was shown for the first time that they defined the basic parameters of the functioning of single ATP-sensitive K+ channels in the human adult ventricular cardiomyocytes membrane. At [K+]o = 140 mM single channel conductance (over the linear part of the I-V relation) reaches 100 pS. The possibility of the existence of these channels' conductance sublevels as well as the cluster character of their localization in sarcolemma is shown. The channel activity demonstrates an obvious run-down with tau at about a minute. The analysed channels possess one open and two closed states.

[Activation of ATP-sensitive K+-channels of cardiomyocytes by endogenous cardiopeptides]. / Aktivatsiia ATF-chuvsvitel'nykh K+-kanalov kardiomiotsitov éndogennymi kardiopeptidami.

The "patch-clamp" method was applied to rat ventricular cardiomyocytes to study the effect of endogenous cardiopeptides (a component of a cardiologic medicine "cardialin") on single K(+)-channel currents. It was noted that ATP-sensitive K(+)-channel activity increased only in the presence of 10(-8)-10(-6) mg.ml-1 peptide solution in the patch-clamp micropipette. To check up the suggestion that G-protein takes part in a direct reaction of K(+)-channel by external ligand we studied the effect of GTP-gamma-S (0.1 mM) in the presence of Mg2+ (1 mM) on the K(+)-channel in "run-down" state in the "inside-out" configuration. It was demonstrated experimentally that reactivation of the ATP-sensitive K(+)-channel develops in the presence of GTP-analogue.

[The characteristics of the reactions of excitable tissue to combined exposure to microwaves and low-intensity ultrasound]. / Osobennosti reaktsii vozbudimoi tkani na kombinirovannoe vozdeistvie mikrovoln i ul'trazvuka nizkoi intensivnosti.

Isolated pacemaker of the frog's heart served as a sensitive model to study the combined action of the low intensity electromagnetic and mechanic factors. Critical energy levels of factors, their relative efficiency in direct action on the cell were estimated. Additive effects of microwaves and ultrasound were examined. The data can be valuable for the further investigations concerning the effects of different nonionizing rays.

[The reaction to hypoxia of ATP-sensitive K+ channels in rat cardiomyocytes]. / Izuchenie reaktsii na gipoksiiu ATF-chuvstvitel'nykh K+-kanalov kardiomiotsitov krysy.

The responses of ATP-sensitive K(+)-channels of the adult rat's cardiomyocytes to simulated hypoxia were found to have an oscillatory character. The degree of its blocking was found to depend on the ATP concentration at the inner side of the membrane. The concentration of the semi-maximal blocking is not more than 20 mcM. The necessity to take into consideration the local intracellular changes of the macroerg content for explanation of the revealed responses, is discussed.