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• W2023446579 abstract "β-Cell-type KATP channels are octamers assembled from Kir6.2/KCNJ11 and SUR1/ABCC8. Adenine nucleotides play a major role in their regulation. Nucleotide binding to Kir6.2 inhibits channel activity, whereas ATP binding/hydrolysis on sulfonylurea receptor 1 (SUR1) opposes inhibition. Segments of the Kir6.2 N terminus are important for open-to-closed transitions, form part of the Kir ATP, sulfonylurea, and phosphoinositide binding sites, and interact with L0, an SUR cytoplasmic loop. Inputs from these elements link to the pore via the interfacial helix, which forms an elbow with the outer pore helix. Mutations that destabilize the interfacial helix increase channel activity, reduce sensitivity to inhibitory ATP and channel inhibitors, glibenclamide and repaglinide, and cause neonatal diabetes. We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle. The substituted channels have increased PO values, decreased sensitivity to inhibitors, and impaired stimulation by phosphoinositides but retain sensitivity to Ba2+-block. The V59G and V59A channels are either not, or poorly, stimulated by phosphoinositides, respectively. Inhibition by sequestrating phosphatidylinositol 4,5-bisphosphate with neomycin and polylysine is reduced in V59A, and abolished in V59G channels. Stimulation by SUR1 is intact, and increasing the concentration of inhibitory ATP restores the sensitivity of Val-59-substituted channels to glibenclamide. The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas. The results suggest the interfacial helix dynamically links inhibitory inputs from the Kir N terminus to the gate and that sulfonylureas stabilize an inhibitory configuration. β-Cell-type KATP channels are octamers assembled from Kir6.2/KCNJ11 and SUR1/ABCC8. Adenine nucleotides play a major role in their regulation. Nucleotide binding to Kir6.2 inhibits channel activity, whereas ATP binding/hydrolysis on sulfonylurea receptor 1 (SUR1) opposes inhibition. Segments of the Kir6.2 N terminus are important for open-to-closed transitions, form part of the Kir ATP, sulfonylurea, and phosphoinositide binding sites, and interact with L0, an SUR cytoplasmic loop. Inputs from these elements link to the pore via the interfacial helix, which forms an elbow with the outer pore helix. Mutations that destabilize the interfacial helix increase channel activity, reduce sensitivity to inhibitory ATP and channel inhibitors, glibenclamide and repaglinide, and cause neonatal diabetes. We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle. The substituted channels have increased PO values, decreased sensitivity to inhibitors, and impaired stimulation by phosphoinositides but retain sensitivity to Ba2+-block. The V59G and V59A channels are either not, or poorly, stimulated by phosphoinositides, respectively. Inhibition by sequestrating phosphatidylinositol 4,5-bisphosphate with neomycin and polylysine is reduced in V59A, and abolished in V59G channels. Stimulation by SUR1 is intact, and increasing the concentration of inhibitory ATP restores the sensitivity of Val-59-substituted channels to glibenclamide. The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas. The results suggest the interfacial helix dynamically links inhibitory inputs from the Kir N terminus to the gate and that sulfonylureas stabilize an inhibitory configuration. ATP-sensitive K+ channels (KATP channels) 2The abbreviations used are: KATP channel, ATP-sensitive K+ channel; DEND syndrome, developmental delay, epilepsy, and neonatal diabetes syndrome; GBC, glibenclamide; Kir, inwardly rectifying potassium channel; PIP2, phosphatidylinositol 4,5-bisphosphate; PO, open probability of single channels; SUR, sulfonylurea receptor. consist of four pore-forming subunits Kir6.x and four sulfonylurea receptor (SURx) subunits, which are members of the ATP-binding cassette (ABC) protein superfamily (1Clement IV, J.P. Kunjilwar K. Gonzalez G. Schwanstecher M. Panten U. Aguilar-Bryan L. Bryan J. Neuron. 1997; 18: 827-838Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). Adenine nucleotides have a balanced action on KATP channels: Mg2+-independent nucleotide binding to Kir6.x closes the channel (2Tucker S.J. Gribble F.M. Zhao C. Trapp S. Ashcroft F.M. Nature. 1997; 387: 179-183Crossref PubMed Scopus (676) Google Scholar, 3Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar, 4Drain P. Li L. Wang J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13953-13958Crossref PubMed Scopus (172) Google Scholar), whereas MgADP binding to, or MgATP hydrolysis by, SURx stimulates channel openings (5Nichols C.G. Shyng S.L. Nestorowicz A. Glaser B. Clement IV, J.P. Gonzalez G. Aguilar-Bryan L. Permutt M.A. Bryan J. Science. 1996; 272: 1785-1787Crossref PubMed Scopus (468) Google Scholar, 6Babenko A.P. J. Biol. Chem. 2008; 283: 8778-8782Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 7Matsuo M. Kimura Y. Ueda K. J. Mol. Cell. Cardiol. 2005; 38: 907-916Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 8Zingman L.V. Alekseev A.E. Bienengraeber M. Hodgson D. Karger A.B. Dzeja P.P. Terzic A. Neuron. 2001; 31: 233-245Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 9Bienengraeber M. Olson T.M. Selivanov V.A. Kathmann E.C. O’Cochlain F. Gao F. Karger A.B. Ballew J.D. Hodgson D.M. Zingman L.V. Pang Y.P. Alekseev A.E. Terzic A. Nat. Genet. 2004; 36: 382-387Crossref PubMed Scopus (295) Google Scholar). These results obtained with recombinant systems give an explanation for the older observation that KATP channels are sensitive to the ratio of ADP to ATP (10Dunne M.J. Petersen O.H. FEBS Lett. 1986; 208: 59-62Crossref PubMed Scopus (184) Google Scholar, 11Misler S. Falke L.C. Gillis K. McDaniel M.L. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7119-7123Crossref PubMed Scopus (232) Google Scholar). There are several subtypes of KATP channels (12Babenko A.P. Aguilar-Bryan L. Bryan J. Annu. Rev. Physiol. 1998; 60: 667-687Crossref PubMed Scopus (482) Google Scholar) that subserve important functions in many tissues (13Seino S. Miki T. Prog. Biophys. Mol. Biol. 2003; 81: 133-176Crossref PubMed Scopus (449) Google Scholar). The Kir6.2/SUR1 neuroendocrine channel in pancreatic β-cells couples insulin secretion to the plasma glucose level. KATP channels determine theβ-cell membrane potential and changes in the levels of ATP and ADP induced by changes in glucose metabolism determine channel activity. Antidiabetic sulfonyl-ureas such as glibenclamide (GBC) bind to SUR1 reducing its stimulatory action on the pore thus inducing channel closure that prompts insulin secretion (14Aguilar-Bryan L. Nichols C.G. Wechsler S.W. Clement IV, J.P. Boyd III, A.E. Gonzalez G. Herrera-Sosa H. Nguy K. Bryan J. Nelson D.A. Science. 1995; 268: 423-426Crossref PubMed Scopus (1282) Google Scholar, 15Proks P. Reimann F. Green N. Gribble F. Ashcroft F.M. Diabetes. 2002; 51: S368-S376Crossref PubMed Google Scholar). Mutations in either subunit can alter the balanced action of adenine nucleotides on KATP channels and result in disorders of insulin secretion (16Ashcroft F.M. J. Clin. Investig. 2005; 115: 2047-2058Crossref PubMed Scopus (455) Google Scholar, 17Aguilar-Bryan L. Bryan J. Endocr. Rev. 2008; 29: 265-291Crossref PubMed Scopus (164) Google Scholar). Loss of channel function is a cause of hyperinsulinemic hypoglycemia (reviewed in Ref. 18Aguilar-Bryan L. Bryan J. Endocr. Rev. 1999; 20: 101-135Crossref PubMed Scopus (620) Google Scholar), whereas mutations that increase channel activity are one cause of neonatal diabetes (reviewed in Ref. 17Aguilar-Bryan L. Bryan J. Endocr. Rev. 2008; 29: 265-291Crossref PubMed Scopus (164) Google Scholar). Mutations in SUR1 have been identified that produce more active channels via increased Mg-nucleotide-dependent stimulation of the pore (6Babenko A.P. J. Biol. Chem. 2008; 283: 8778-8782Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 19Babenko A.P. Polak M. Cave H. Busiah K. Czernichow P. Scharfmann R. Bryan J. Aguilar-Bryan L. Vaxillaire M. Froguel P. N. Engl. J. Med. 2006; 355: 456-466Crossref PubMed Scopus (550) Google Scholar, 20Flanagan S.E. Patch A.M. Mackay D.J. Edghill E.L. Gloyn A.L. Robinson D. Shield J.P. Temple K. Ellard S. Hattersley A.T. Diabetes. 2007; 56: 1930-1937Crossref PubMed Scopus (279) Google Scholar; reviewed in Ref. 17Aguilar-Bryan L. Bryan J. Endocr. Rev. 2008; 29: 265-291Crossref PubMed Scopus (164) Google Scholar). Mutations in Kir6.2 have been identified in which the ability of ATP to close the channel is reduced due to a decreased affinity for inhibitory ATP or to an increased stability of the open state in the absence of ATP (16Ashcroft F.M. J. Clin. Investig. 2005; 115: 2047-2058Crossref PubMed Scopus (455) Google Scholar, 21Gloyn A.L. Pearson E.R. Antcliff J.F. Proks P. Bruining G.J. Slingerland A.S. Howard N. Srinivasan S. Silva J.M. Molnes J. Edghill E.L. Frayling T.M. Temple I.K. Mackay D. Shield J.P. Sumnik Z. van Rhijn A. Wales J.K. Clark P. Gorman S. Aisenberg J. Ellard S. Njolstad P.R. Ashcroft F.M. Hattersley A.T. N. Engl. J. Med. 2004; 350: 1838-1849Crossref PubMed Scopus (975) Google Scholar, 22Koster J.C. Remedi M.S. Dao C. Nichols C.G. Diabetes. 2005; 54: 2645-2654Crossref PubMed Scopus (87) Google Scholar). In patients carrying one copy of these “gain of function” mutations the balanced action of adenine nucleotides is altered and the resulting increase in channel activity leads to β-cell hyperpolarization and the decrease in insulin secretion that causes (transient or permanent) neonatal diabetes. KATP channel subunits are found in neurons (Kir6.2 with SUR1), in striated muscle (Kir6.2 with SUR2A), and in some smooth muscle (Kir6.1 with SUR2B). Some Kir6.2 mutations result in hyperactive channels that produce more severe syndromic phenotypes that include muscle weakness, developmental delay, epilepsy, and neonatal diabetes, termed the DEND syndrome (16Ashcroft F.M. J. Clin. Investig. 2005; 115: 2047-2058Crossref PubMed Scopus (455) Google Scholar, 21Gloyn A.L. Pearson E.R. Antcliff J.F. Proks P. Bruining G.J. Slingerland A.S. Howard N. Srinivasan S. Silva J.M. Molnes J. Edghill E.L. Frayling T.M. Temple I.K. Mackay D. Shield J.P. Sumnik Z. van Rhijn A. Wales J.K. Clark P. Gorman S. Aisenberg J. Ellard S. Njolstad P.R. Ashcroft F.M. Hattersley A.T. N. Engl. J. Med. 2004; 350: 1838-1849Crossref PubMed Scopus (975) Google Scholar, 23Hattersley A.T. Ashcroft F.M. Diabetes. 2005; 54: 2503-2513Crossref PubMed Scopus (370) Google Scholar, 24Koster J.C. Permutt M.A. Nichols C.G. Diabetes. 2005; 54: 3065-3072Crossref PubMed Scopus (131) Google Scholar, 25Flanagan S.E. Edghill E.L. Gloyn A.L. Ellard S. Hattersley A.T. Diabetologia. 2006; 49: 1190-1197Crossref PubMed Scopus (194) Google Scholar). One DEND mutation, V59G, is in the slide or interfacial helix of Kir6.2 (21Gloyn A.L. Pearson E.R. Antcliff J.F. Proks P. Bruining G.J. Slingerland A.S. Howard N. Srinivasan S. Silva J.M. Molnes J. Edghill E.L. Frayling T.M. Temple I.K. Mackay D. Shield J.P. Sumnik Z. van Rhijn A. Wales J.K. Clark P. Gorman S. Aisenberg J. Ellard S. Njolstad P.R. Ashcroft F.M. Hattersley A.T. N. Engl. J. Med. 2004; 350: 1838-1849Crossref PubMed Scopus (975) Google Scholar), an amphipathic stretch of 13 amino acids (residues 54–66) that lies at the membrane-cytosol interface and is assumed to be important in the mechanics of channel gating (26Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; 300: 1922-1926Crossref PubMed Scopus (736) Google Scholar, 27Nishida M. Cadene M. Chait B.T. MacKinnon R. EMBO J. 2007; 26: 4005-4015Crossref PubMed Scopus (257) Google Scholar, 28Antcliff J.F. Haider S. Proks P. Sansom M.S. Ashcroft F.M. EMBO J. 2005; 24: 229-239Crossref PubMed Scopus (168) Google Scholar). In intact Xenopus oocytes, homozygous V59G channels are essentially open (29Proks P. Antcliff J.F. Lippiat J. Gloyn A.L. Hattersley A.T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17539-17544Crossref PubMed Scopus (209) Google Scholar) and in isolated patches, 3 mm MgATP produces only a 10% block; in addition, the sensitivity of heterozygous channels to tolbutamide is strongly reduced (29Proks P. Antcliff J.F. Lippiat J. Gloyn A.L. Hattersley A.T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17539-17544Crossref PubMed Scopus (209) Google Scholar, 30Proks P. Girard C. Ashcroft F.M. Hum. Mol. Genet. 2005; 14: 2717-2726Crossref PubMed Scopus (69) Google Scholar). In experiments at the single channel level in the absence of ATP, homozygous V59G channels exhibit a high open probability (PO = 0.83 versus 0.53 for the wild type (29Proks P. Antcliff J.F. Lippiat J. Gloyn A.L. Hattersley A.T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17539-17544Crossref PubMed Scopus (209) Google Scholar)). The reduced sensitivity of V59G channels to inhibition by ATP and tolbutamide has been attributed to the higher open probability, because both compounds stabilize a long lived interburst closed state (31Trapp S. Proks P. Tucker S.J. Ashcroft F.M. J. Gen. Physiol. 1998; 112: 333-349Crossref PubMed Scopus (150) Google Scholar, 32Enkvetchakul D. Loussouarn G. Makhina E. Shyng S.L. Nichols C.G. Biophys. J. 2000; 78: 2334-2348Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 33Koster J.C. Sha Q. Shyng S. Nichols C.G. J. Physiol. 1999; 515: 19-30Crossref PubMed Scopus (86) Google Scholar, 34Babenko A.P. Bryan J. J. Biol. Chem. 2001; 276: 49083-49092Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In addition, the V59G substitution reduces the surface expression of the mutant channel to ∼20% of wild type (35Lin C.W. Lin Y.W. Yan F.F. Casey J. Kochhar M. Pratt E.B. Shyng S.L. Diabetes. 2006; 55: 1738-1746Crossref PubMed Scopus (35) Google Scholar). Two other substitutions of Val-59 occur, V59M (21Gloyn A.L. Pearson E.R. Antcliff J.F. Proks P. Bruining G.J. Slingerland A.S. Howard N. Srinivasan S. Silva J.M. Molnes J. Edghill E.L. Frayling T.M. Temple I.K. Mackay D. Shield J.P. Sumnik Z. van Rhijn A. Wales J.K. Clark P. Gorman S. Aisenberg J. Ellard S. Njolstad P.R. Ashcroft F.M. Hattersley A.T. N. Engl. J. Med. 2004; 350: 1838-1849Crossref PubMed Scopus (975) Google Scholar) and V59A, 3L. Philipson and G. Bell, personal communication. which produce an “intermediate” DEND phenotype. The V59M channel has been extensively characterized (22Koster J.C. Remedi M.S. Dao C. Nichols C.G. Diabetes. 2005; 54: 2645-2654Crossref PubMed Scopus (87) Google Scholar, 29Proks P. Antcliff J.F. Lippiat J. Gloyn A.L. Hattersley A.T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17539-17544Crossref PubMed Scopus (209) Google Scholar, 30Proks P. Girard C. Ashcroft F.M. Hum. Mol. Genet. 2005; 14: 2717-2726Crossref PubMed Scopus (69) Google Scholar), and the properties of the recently identified V59A channel have not been established. Homology models imply the interfacial helix could serve to transmit conformational changes from the regulatory subunit to the outer transmembrane (M1) helices of Kir6.1 or 6.2 pores and thus affect gating. The nature of these conformational changes is uncertain, but the first N-terminal residues of Kir6.2 are known to be important for transitions from the open to the long lived closed state. Deletions of 5–35 residues from Kir6.1 and Kir6.2 produce highly active channels, PO > 0.9 (36Babenko A.P. Bryan J. J. Biol. Chem. 2002; 277: 43997-44004Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 37Babenko A.P. Gonzalez G. Bryan J. Biochem. Biophys. Res. Commun. 1999; 255: 231-238Crossref PubMed Scopus (68) Google Scholar), with reduced sensitivity to sulfonylureas and ATP (3Babenko A.P. Gonzalez G. Aguilar-Bryan L. Bryan J. FEBS Lett. 1999; 445: 131-136Crossref PubMed Scopus (51) Google Scholar, 33Koster J.C. Sha Q. Shyng S. Nichols C.G. J. Physiol. 1999; 515: 19-30Crossref PubMed Scopus (86) Google Scholar, 37Babenko A.P. Gonzalez G. Bryan J. Biochem. Biophys. Res. Commun. 1999; 255: 231-238Crossref PubMed Scopus (68) Google Scholar, 38Reimann F. Tucker S.J. Proks P. Ashcroft F.M. J. Physiol. 1999; 518: 325-336Crossref PubMed Scopus (88) Google Scholar), similar to those seen in the V59G channels. We suggest that the N terminus of Kir6.x is an inhibitory element that interacts with parts of SUR1, specifically with the L0 linker, to facilitate the transition to the closed state and thus to restrict channel openings (39Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The observation that soluble, Kir N-terminal-like peptides can reduce the PO of ΔNKir6.1/SUR1 and ΔNKir6.2/SUR1 channels supports this idea (36Babenko A.P. Bryan J. J. Biol. Chem. 2002; 277: 43997-44004Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The interfacial helix is the physical connection between the proximal N terminus and the outer M1 helix. We propose that substitutions (e.g. glycine, methionine, and alanine) for valine at position 59 can introduce flexibility into the V59G helix that impairs the inhibitory action of the proximal N terminus. To support this hypothesis, we sought to characterize the V59G mutation further, compare it with a V59A substitution and the analogous substitution, I60G, in Kir6.1 the pore-forming subunit of the vascular KATP channel. This channel is composed of Kir6.1 and SUR2B (40Yamada M. Isomoto S. Matsumoto S. Kondo C. Shindo T. Horio Y. Kurachi Y. J. Physiol. 1997; 499: 715-720Crossref PubMed Scopus (342) Google Scholar), however, for comparison we examined the I60G/SUR1 channel. Kir6.1 and 6.2 are 71% homologous overall, and the amino acids in the interfacial helix are identical except Val-59 is replaced by Ile-60 in Kir6.1. The Kir6.1 and Kir6.2 channels differ in their unitary conductance (∼36 versus 66 pS for Kir6.1 versus Kir6.2, respectively (41Takano M. Xie L.H. Otani H. Horie M. J. Physiol. 1998; 512: 395-406Crossref PubMed Scopus (35) Google Scholar)). The channels also differ in their gating by nucleotides in isolated patches: Kir6.1/SUR1 channels require strong activating conditions, whereas Kir6.2/SUR1 channels are open in nucleotide-free solutions (42Beech D.J. Zhang H. Nakao K. Bolton T.B. Br. J. Pharmacol. 1993; 110: 573-582Crossref PubMed Scopus (170) Google Scholar, 43Satoh E. Yamada M. Kondo C. Repunte V.P. Horio Y. Iijima T. Kurachi Y. J. Physiol. 1998; 511: 663-674Crossref PubMed Scopus (54) Google Scholar). The Kir6.1-based channels have been reported either to have very low (41Takano M. Xie L.H. Otani H. Horie M. J. Physiol. 1998; 512: 395-406Crossref PubMed Scopus (35) Google Scholar, 43Satoh E. Yamada M. Kondo C. Repunte V.P. Horio Y. Iijima T. Kurachi Y. J. Physiol. 1998; 511: 663-674Crossref PubMed Scopus (54) Google Scholar) or equivalent (34Babenko A.P. Bryan J. J. Biol. Chem. 2001; 276: 49083-49092Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) sensitivity to inhibitory ATP when compared with Kir6.2-based channels. In view of these differences in gating on one hand and of the similarity in amino acid sequence around the mutation on the other it was of interest to compare the V59G and I60G/SUR1 KATP channels. Structure Prediction-To examine the effect of the mutations on the secondary structure of the interfacial helix of Kir6.x (Kir6.1: amino acids 55–67, Kir6.2: 54–66), the following programs were used: Predator (version 5a), JPRED, PSIPRED, nnpredict, and PhD (see Ref. 44Rost B. Methods Biochem. Anal. 2003; 44: 559-587PubMed Google Scholar for references). Molecular Biology, Cell Culture, and Transfection-Human Kir6.1, Kir6.2, and SUR1 cDNAs were subcloned into the pcDNA3.1 vector. A Myc epitope was introduced into human Kir6.2 after leucine 100 by ligating complementary oligonucleotides with appropriate overhangs; this modification did not affect the functional properties of the protein (39Babenko A.P. Bryan J. J. Biol. Chem. 2003; 278: 41577-41580Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The V59G, V59A, and I60G mutations were introduced into Kir6.2 and Kir6.1, respectively, using the QuikChange II XL site-directed mutagenesis kit (Stratagene). The mutations were verified by sequencing the relevant DNA region (V59G) or the whole coding region (I60G and V59A). HEK 293 cells were cultured in minimum essential medium containing glutamine and supplemented with 10% fetal bovine serum and 20 μg/ml gentamycin as described (45Hambrock A. Loffler-Walz C. Russ U. Lange U. Quast U. Mol. Pharmacol. 2001; 60: 190-199Crossref PubMed Scopus (53) Google Scholar). Cells were transfected with wild-type or mutant Kir6.x and SUR1 at a molar ratio of 1:1 using Lipofectamine 2000 and Opti-MEM (Invitrogen) according to the manufacturer’s instructions (46Hambrock A. Loffler-Walz C. Kurachi Y. Quast U. Br. J. Pharmacol. 1998; 125: 577-583Crossref PubMed Scopus (66) Google Scholar). The pEGFP-C1 vector (Clontech, Palo Alto, CA), encoding green fluorescent protein, was added for identification of transfected cells. Patch Clamp Experiments-Patch clamp experiments in the whole cell configuration were performed at 37 °C as described by Russ et al. (47Russ U. Hambrock A. Artunc F. Loffler-Walz C. Horio Y. Kurachi Y. Quast U. Mol. Pharmacol. 1999; 56: 955-961Crossref PubMed Scopus (44) Google Scholar). The bath was filled with (in mm): NaCl, 142; KCl, 2.8; MgCl2, 1; CaCl2, 1; d(+)-glucose, 11; HEPES, 10; pH 7.4. Patch pipettes were filled with (in mm) potassium glutamate, 132; NaCl, 10; MgCl2, 2; HEPES, 10; EGTA, 1; Na2ATP, 1; and Na2GDP, 0.3 at pH 7.2 such that [Mg2+]free was ∼0.85 mm and that there was a balance between inhibition and activation. Pipettes had a resistance of 3–5 MΩ. Cells were clamped at –60 mV. To determine the reversal potential of the currents, square pulses, 0.5-s duration, ranging from –110 to +10 mV in 20-mV steps were applied every 12 s. Recordings in which the reversal potential deviated from ∼–90 mV were rejected. For experiments in the cell-attached and inside-out configuration, pipettes with a larger diameter were used (resistance of 1.0–1.5 MΩ). Experiments were performed at 22 °C. Pipette and bath were filled with a high K+-Ringer solution containing (in mm) KCl, 142; NaCl, 2.8; MgCl2, 1; CaCl2, 1; d(+)-glucose, 11; HEPES, 10; titrated to pH 7.4 with NaOH. In the inside-out mode and after patch excision, the pipette was moved in front of a pipe filled with a high K+ buffer containing (in mm) KCl, 142; MgCl2, 0.7–30.7 (according to the nucleotides added); d(+)-glucose, 11; Na2ATP, 0–30; EGTA, 0.1 (0 when adding Ba2+); HEPES, 10; titrated to pH 7.2 with NaOH at 22 °C and containing the channel modulators of interest. For experiments in Mg2+-free buffer, MgCl2 was omitted and EGTA was replaced by 1 mm EDTA. In a series of experiments using high ATP concentrations (10 and 30 mm), NaCl was added to the bath and pipe solutions with lower ATP concentration to minimize differences in osmolarity. Patches were generally clamped at –50 mV except for examination of the Ba2+-induced block in the inside-out configuration. Ba2+ applied to the inside of the patch produced only a slight block at –50 mV (because it was flushed out of the pore by the inward K+ current at negative voltage), whereas at +50 mV a complete block was achieved (as Ba2+ was dragged into the pore by the outward current). Some inhibitors (e.g. neomycin) were tested at both –50 and +50 mV. In experiments with the V59G/SUR1 channel in the inside-out configuration and at symmetrical high K+ solution, the leak current was determined as the outward current remaining in the presence of 1 mm Ba2+ at +50 mV, and it was assumed that, at –50 mV, the leak current was of the same magnitude (with inverted sign), thus determining the zero current level. Data were filtered at 0.2 kHz and sampled at 1 kHz. The open probability (PO) of the V59A channel was determined in the inside-out configuration using Sylgard-coated pipettes. Recordings from patches with one channel were filtered at 2.5 kHz and sampled at 5 kHz. Amplitude histograms were generated using PulseTools (Heka, Lambrecht, Germany) and analyzed by fitting a superposition of 2 Gaussian distributions to the data. [3H]GBC Competition Experiments-Binding experiments were performed in intact cells at 37 °C as described by Hambrock et al. (45Hambrock A. Loffler-Walz C. Russ U. Lange U. Quast U. Mol. Pharmacol. 2001; 60: 190-199Crossref PubMed Scopus (53) Google Scholar) using an incubation buffer containing (in mm): NaCl, 129; KCl, 5; MgCl2, 1.2; CaCl2, 1.25; d(+)-glucose, 11; NaHCO3, 5; HEPES, 10 at pH 7.4. [3H]GBC was 1–2 nm; nonspecific binding was determined in the presence of 100 nm GBC and was ∼10% of total binding. Data Analysis and Statistics-Channel inhibition and equilibrium binding inhibition curves were analyzed according to the Hill equation,y=100-A(1+10nH(pX-pIC50))Eq. 1 as described before (45Hambrock A. Loffler-Walz C. Russ U. Lange U. Quast U. Mol. Pharmacol. 2001; 60: 190-199Crossref PubMed Scopus (53) Google Scholar) with y denoting the current or total binding, A the maximum inhibition (amplitude or extent of specific binding), nH the Hill coefficient, and x the inhibitor concentration with pX = –log X and pIC50 = –log IC50. In the text, the IC50 values with their 95% confidence interval are given. IC50 values are lognormally distributed (48Christopoulos A. Trends Pharmacol. Sci. 1998; 19: 351-357Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar); therefore the corresponding pIC50 values were compared by using the Student t-test after the data had passed the normality and equal variance tests using the program SigmaStat 3.1 (SPSS Science, Chicago, IL). Materials-The reagents and media used for cell culture and transfection were from Invitrogen, the other chemicals, including nucleotides, were from Sigma. Glibenclamide, tolbutamide, and diazoxide were purchased from Sigma; repaglinide was a kind gift from Novo Nordisk (Bagsvaerd, Denmark). The KATP channel modulators were dissolved in DMSO/ethanol (50/50, v/v) and further diluted with the same solvent or with incubation buffer (final solvent concentration in the assays, <1%). [3H]GBC (specific activity, 1.85 TBq/mmol) was purchased from Perkin-Elmer Life Sciences. Poly-d-lysine-HBr (mean molecular weight 41,400; chain length ∼200) was from Sigma. Structure Prediction-Glycine has a lower helix propensity than valine (49Chou P.Y. Fasman G.D. Annu. Rev. Biochem. 1978; 47: 251-276Crossref PubMed Scopus (2336) Google Scholar), and two, Predator and nnpredict, out of five protein secondary structure programs predicted that the V59G substitution will destabilize and thus break the interfacial helix. The PSIPRED and PhD programs predicted no structural change with the V59G mutation; JPRED did not recognize the helix structure. The same structural predictions were obtained for the I60G substitution in Kir6.1. Alanine and methionine have a greater helix propensity on the Chou-Fasman scale (49Chou P.Y. Fasman G.D. Annu. Rev. Biochem. 1978; 47: 251-276Crossref PubMed Scopus (2336) Google Scholar) than glycine, are not expected to interrupt the interfacial helix, but may change its flexibility. Basic Observations on Kir6.2 Channels-Fig. 1 shows basic characteristics of the wild-type and Val-59-substituted channels determined in the whole cell configuration. After breaking into a cell expressing wild-type channels, a current developed during cell dialysis with a nucleotide containing activating solution (Fig. 1a). The current was totally inhibited by glibenclamide (GBC, 0.1 μm), indicating it goes through KATP channels. In contrast, in cells expressing the Val-59-substituted channels, a current was present immediately upon breaking into the cell indicating channels were open prior to dialysis (Fig. 1, b and c). In agreement with their reported reduced sensitivity to sulfonylureas (29Proks P. Antcliff J.F. Lippiat J. Gloyn A.L. Hattersley A.T. Ashcroft F.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 17539-17544Crossref PubMed Scopus (209) Google Scholar) the V59G currents were not affected by GBC (1 μm) (Fig. 1b; n = 12). The V59A channels display an intermediate sensitivity to 1 μm glibenclamide, ∼40% inhibition. Both of the Val-59-subsitituted channels are blocked by a high concentration of Ba2+ (1 mm). Fig. 2 shows there is no significant difference in Ba2+ sensitivity between the wild-type and V59G channels.FIGURE 2Concentration-dependent inhibition of V59G and wild-type Kir6.2/SUR1 channels by Ba2+. Experiments were performed using the inside-out configuration of the patch clamp technique at +50 mV (see “Experimental Procedures”). Data are means of eight and six patches for the mutant and wild-type channels, respectively, and curve analysis with the Hill coefficient 1.0 gave pIC50 values of 5.38 ± 0.02 and 5.45 ± 0.07, respectively (not different). The inset shows a typical experiment with V59G channels. The zero current level was determined in the presence of Ba2+ (1 mm) and is given by the dotted line. There was a slight channel rundown; Ba2+ was applied via a pipe to the inside of the patch.View Large Image Figure ViewerDown" @default.
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• W2023446579 title "Analysis of Two KCNJ11 Neonatal Diabetes Mutations, V59G and V59A, and the Analogous KCNJ8 I60G Substitution" @default.
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