ABCMdb

ABCC8 p.Cys717Ser

Predicted by SNAP2:	A: N (57%), D: D (91%), E: D (85%), F: D (85%), G: D (85%), H: D (91%), I: D (85%), K: D (91%), L: D (91%), M: D (80%), N: D (91%), P: D (91%), Q: D (85%), R: D (91%), S: N (53%), T: D (80%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] Nitric oxide activates ATP-sensitive potassium cha... Mol Pain. 2009 Mar 14;5:12. Kawano T, Zoga V, Kimura M, Liang MY, Wu HE, Gemes G, McCallum JB, Kwok WM, Hogan QH, Sarantopoulos CD
Nitric oxide activates ATP-sensitive potassium channels in mammalian sensory neurons: action by direct S-nitrosylation.
Mol Pain. 2009 Mar 14;5:12., [PMID:19284878]

Abstract [show]
BACKGROUND: ATP-sensitive potassium (KATP) channels in neurons regulate excitability, neurotransmitter release and mediate protection from cell-death. Furthermore, activation of KATP channels is suppressed in DRG neurons after painful-like nerve injury. NO-dependent mechanisms modulate both KATP channels and participate in the pathophysiology and pharmacology of neuropathic pain. Therefore, we investigated NO modulation of KATP channels in control and axotomized DRG neurons. RESULTS: Cell-attached and cell-free recordings of KATP currents in large DRG neurons from control rats (sham surgery, SS) revealed activation of KATP channels by NO exogenously released by the NO donor SNAP, through decreased sensitivity to [ATP]i. This NO-induced KATP channel activation was not altered in ganglia from animals that demonstrated sustained hyperalgesia-type response to nociceptive stimulation following spinal nerve ligation. However, baseline opening of KATP channels and their activation induced by metabolic inhibition was suppressed by axotomy. Failure to block the NO-mediated amplification of KATP currents with specific inhibitors of sGC and PKG indicated that the classical sGC/cGMP/PKG signaling pathway was not involved in the activation by SNAP. NO-induced activation of KATP channels remained intact in cell-free patches, was reversed by DTT, a thiol-reducing agent, and prevented by NEM, a thiol-alkylating agent. Other findings indicated that the mechanisms by which NO activates KATP channels involve direct S-nitrosylation of cysteine residues in the SUR1 subunit. Specifically, current through recombinant wild-type SUR1/Kir6.2 channels expressed in COS7 cells was activated by NO, but channels formed only from truncated isoform Kir6.2 subunits without SUR1 subunits were insensitive to NO. Further, mutagenesis of SUR1 indicated that NO-induced KATP channel activation involves interaction of NO with residues in the NBD1 of the SUR1 subunit. CONCLUSION: NO activates KATP channels in large DRG neurons via direct S-nitrosylation of cysteine residues in the SUR1 subunit. The capacity of NO to activate KATP channels via this mechanism remains intact even after spinal nerve ligation, thus providing opportunities for selective pharmacological enhancement of KATP current even after decrease of this current by painful-like nerve injury.

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No. Sentence Comment
189 We therefore focused on this cysteine residue, and examined the effect of mutating C717 to serine (C717S) on the sensitivity of the cloned SUR1/Kir6.2 channel to NO. Login to comment
190 A patch containing recombinant SUR1-C717S/Kir6.2 channels, also showed marked current increases when excised into a low [ATP]i (0.1 μM) solution (Figure 9A). Login to comment
192 The ATP-sensitivity of SUR1-C717S/Kir6.2 channels (IC50 = 13.1 μM, n = 7) was not significantly different from that of wild type SUR1/Kir6.2 channels (IC50 = 11.6 μM, n = 7). Login to comment
193 Similar to the response of wild type SUR1/Kir6.2 channels, bath application of 1 mM SNAP significantly activated the SUR1-C717S/Kir6.2 channel in the presence of 1 mM [ATP]i. However SNAP was significantly less potent in activating the SUR1-C717S/Kir6.2 channel compared with the wild-type SUR1/Kir6.2 channel (n = 8, p < 0.02 vs. wild type; Figure 9C). Login to comment
194 SNAP-induced SUR1-C717S/Kir6.2 channel activation was reversed by DTT (5 mM) (n = 7). Login to comment
195 In addition to C717S, we further examined the activating effects of NO after mutating the cysteine residues within NBD1 and NBD2 to serine, including those cysteine residues adjacent to C717 within NBD1 (C806S), and all three cysteine residues within NBD2 (C1378, C1446, C1491). Login to comment
197 Among these mutants, only SUR1-C717S/Kir6.2 channels significantly affected NO-sensitivity compared with wild type SUR1/Kir6.2 channels. Login to comment
213 Figure 9 (see legend on next page) A B SUR1-C717/Kir6.2 Wild type SUR1/Kir6.2 SUR1-C717S/Kir6.2 Relativechannelactivity ATP 1 μM ATP 1 mM 5 pA 200 ms [ATP]i (μM) C SNAP ATP 1 mM SNAP + DTT ATP 1 mM (ATP 1 mM) SNAP (+) Relative channel activity wt SUR1 SUR1-C717S SUR1-C806S SUR1-C1378S SUR1-C1446S SUR1-C1491S wt SUR1 SUR1-C717S SUR1-C806S SUR1-C1378S SUR1-C1446S SUR1-C1491S control D SUR1-C717/Kir6.2 5 pA 200 ms * Although the sGC/cGMP/PKG pathway mediates NO regulation of KATP channels in cardiomyocytes, vascular and non-vascular smooth muscle cells, and pancreatic β cells [52-56], this is not the case in DRG neurons. Login to comment
235 (A) Representative traces of currents via SUR1-C717S/Kir6.2 channels in inside-out patches at -60 mV holding potential. Login to comment
236 Bath application of ATP (1 mM) inhibited SUR1-C717S/Kir6.2 channels. Login to comment
237 Arrows indicate closed channel state. (B) Concentration-response relationship curves between [ATP]i and relative NPo values for wild-type SUR1/Kir6.2 or SUR1-C717S/Kir6.2 channels. Login to comment
240 (C) Representative current traces of SUR1-C717S/ Kir6.2 channel currents in inside-out patches at -60 mV holding potential. In the presence of 1 mM [ATP]i, bath application of SNAP (1 mM) activated SUR1-C717S/Kir6.2 channel, but less than wild-type SUR1/Kir6.2 channels. Login to comment
301 Point mutation of cysteine at position 717 (C717), C806, C1378, C1446, and C1491 within SUR1 to serine (C717S, C806S, C1378S, C1446S, and C1491S) was performed by using the Site-Directed Mutagenesis system (Invitrogen Corp.). Login to comment
191 We therefore focused on this cysteine residue, and examined the effect of mutating C717 to serine (C717S) on the sensitivity of the cloned SUR1/Kir6.2 channel to NO. Login to comment
196 However SNAP was significantly less potent in activating the SUR1-C717S/Kir6.2 channel compared with the wild-type SUR1/Kir6.2 channel (n = 8, p < 0.02 vs. wild type; Figure 9C). Login to comment
198 In addition to C717S, we further examined the activating effects of NO after mutating the cysteine residues within NBD1 and NBD2 to serine, including those cysteine residues adjacent to C717 within NBD1 (C806S), and all three cysteine residues within NBD2 (C1378, C1446, C1491). Login to comment
200 Among these mutants, only SUR1-C717S/Kir6.2 channels significantly affected NO-sensitivity compared with wild type SUR1/Kir6.2 channels. Login to comment
216 of 20 (page number not for citation purposes) Figure 9 (see legend on next page) A B SUR1-C717/Kir6.2 Wild type SUR1/Kir6.2 SUR1-C717S/Kir6.2 Relative channel activity ATP 1 bc;M ATP 1 mM 5 pA 200 ms [ATP]i (bc;M) C SNAP ATP 1 mM SNAP + DTT ATP 1 mM (ATP 1 mM) SNAP (+) Relative channel activity wt SUR1 SUR1-C717S SUR1-C806S SUR1-C1378S SUR1-C1446S SUR1-C1491S wt SUR1 SUR1-C717S SUR1-C806S SUR1-C1378S SUR1-C1446S SUR1-C1491S control D SUR1-C717/Kir6.2 5 pA 200 ms * of 20 (page number not for citation purposes) Although the sGC/cGMP/PKG pathway mediates NO regulation of KATP channels in cardiomyocytes, vascular and non-vascular smooth muscle cells, and pancreatic b2; cells [52-56], this is not the case in DRG neurons. Login to comment
239 (A) Representative traces of currents via SUR1-C717S/Kir6.2 channels in inside-out patches at -60 mV holding potential. Login to comment
241 Arrows indicate closed channel state. (B) Concentration-response relationship curves between [ATP]i and relative NPo values for wild-type SUR1/Kir6.2 or SUR1-C717S/Kir6.2 channels. Login to comment
244 (C) Representative current traces of SUR1-C717S/ Kir6.2 channel currents in inside-out patches at -60 mV holding potential. In the presence of 1 mM [ATP]i, bath application of SNAP (1 mM) activated SUR1-C717S/Kir6.2 channel, but less than wild-type SUR1/Kir6.2 channels. Login to comment
305 Point mutation of cysteine at position 717 (C717), C806, C1378, C1446, and C1491 within SUR1 to serine (C717S, C806S, C1378S, C1446S, and C1491S) was performed by using the Site-Directed Mutagenesis system (Invitrogen Corp.). Login to comment

[hide] NEM modification prevents high-affinity ATP bindin... FEBS Lett. 1999 Sep 24;458(3):292-4. Matsuo M, Tucker SJ, Ashcroft FM, Amachi T, Ueda K
NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1.
FEBS Lett. 1999 Sep 24;458(3):292-4., [PMID:10570926]

Abstract [show]
Pancreatic beta-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 microM 8-azido-[alpha-32P]ATP or 8-azido-[gamma-32P]ATP was inhibited by NEM with Ki of 1.8 microM and 2.4 microM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 microM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1.

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No. Sentence Comment
3 However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 WWM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1. Login to comment
27 The C717S and K1385M mutations used in this study were made in rat SUR1. Login to comment
58 We therefore examined the e¡ects of NEM on 8-azido-ATP binding to a mutant form of SUR1, in which the cysteine residue within the Walker A motif of NBF1 was replaced with serine (C717S). Login to comment
59 Membranes containing equal amounts of the wild-type and the C717S mutant form of SUR1 were treated with 100 WM NEM, and photoa¤nity labeling with 5 WM 8-azido-[Q-32 P]- ATP was then examined (Fig. 3). Login to comment
60 Both wild-type and C717S SUR1 were photoa¤nity labeled to the same extent in the absence of NEM. Login to comment
61 However, by contrast to wild-type SUR1, photoa¤nity labeling of C717S SUR1 was una¡ected by pretreatment with NEM. Login to comment
73 The cysteine residue within the Walker A motif of NBF of SUR1 was replaced with serine in the C717S mutant form. Login to comment
77 The inhibition of this high-a¤nity 8-azido-ATP binding to SUR1 by NEM and the lack of inhibition found with the C717S mutation now con'rms that NBF1 is the high-a¤nity ATP binding site identi'ed on SUR1. Login to comment