ABCMdb

PMID: 19284878

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., [PubMed]

Sentences

No. Mutations Sentence Comment

189 ABCC8 p.Cys717Ser 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 ABCC8 p.Cys717Ser 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 191 ABCC8 p.Cys717Ser 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 192 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser 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 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser 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 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser SNAP-induced SUR1-C717S/Kir6.2 channel activation was reversed by DTT (5 mM) (n = 7). Login to comment 195 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser 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 196 ABCC8 p.Cys717Ser 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 197 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser Among these mutants, only SUR1-C717S/Kir6.2 channels significantly affected NO-sensitivity compared with wild type SUR1/Kir6.2 channels. Login to comment 198 ABCC8 p.Cys717Ser 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 ABCC8 p.Cys717Ser 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 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser 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 216 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser 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 235 ABCC8 p.Cys717Ser (A) Representative traces of currents via SUR1-C717S/Kir6.2 channels in inside-out patches at -60 mV holding potential. Login to comment 236 ABCC8 p.Cys717Ser Bath application of ATP (1 mM) inhibited SUR1-C717S/Kir6.2 channels. Login to comment 237 ABCC8 p.Cys717Ser 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 239 ABCC8 p.Cys717Ser (A) Representative traces of currents via SUR1-C717S/Kir6.2 channels in inside-out patches at -60 mV holding potential. Login to comment 240 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser (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 241 ABCC8 p.Cys717Ser 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 ABCC8 p.Cys717Ser ABCC8 p.Cys717Ser (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 ABCC8 p.Cys717Ser 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 305 ABCC8 p.Cys717Ser 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