ABCMdb

PMID: 10469651

Beguin P, Nagashima K, Nishimura M, Gonoi T, Seino S
PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.
EMBO J. 1999 Sep 1;18(17):4722-32., [PubMed]

Sentences

No. Mutations Sentence Comment

65 ABCC8 p.Ser1500Ala ABCC8 p.Ser1446Ala Both the S1500A and S1446A mutants were phosphorylated to the same extent as the wild type (Figure 3B, compare lane 4 with lane 9 or 11). Login to comment 66 ABCC8 p.Ser1500Ala ABCC8 p.Ser1446Ala ABCC8 p.Ser1571Ala Both the S1500A and S1446A mutants were phosphorylated to the same extent as the wild type (Figure 3B, compare lane 4 with lane 9 or 11). Login to comment 67 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Asp In contrast, the phosphorylation in the S1571A mutant in response to PKA was completely abolished (Figure 3B, compare lane 4 with 7). Login to comment 68 ABCC8 p.Ser1571Asp PKA-mediated phosphorylation of human SUR1 was also abolished by the substitution of serine-1571 with aspartic acid (unpublished data). Login to comment 76 ABCC8 p.Ser1571Ala PKA-mediated phosphorylation in the human b2;-cell KATP channel in intact cells Although our data indicate clearly that phosphorylation occurs at specific sites in KATP channels, to determine if these sites are phosphorylated in intact cells, we transiently transfected COS-1 cells with either human wt Kir6.2 or mutant Kir6.2 S372A cDNA together with either human wt SUR1 or the mutant SUR1 S1571A cDNA, and tested their ability to be phosphorylated in intact cells after PKA stimulation. Login to comment 77 ABCC8 p.Ser1571Ala PKA-mediated phosphorylation in the human β-cell KATP channel in intact cells Although our data indicate clearly that phosphorylation occurs at specific sites in KATP channels, to determine if these sites are phosphorylated in intact cells, we transiently transfected COS-1 cells with either human wt Kir6.2 or mutant Kir6.2 S372A cDNA together with either human wt SUR1 or the mutant SUR1 S1571A cDNA, and tested their ability to be phosphorylated in intact cells after PKA stimulation. Login to comment 83 ABCC8 p.Ser1571Ala In addition, mutation of the PKA site (SUR1 S1571A) did not result in any significant decrease in phosphorylation in the basal state (Figure 4C, lane 7), and the PKA inhibitor H-89 did not reduce this basal phosphorylation (unpublished data), indicating that basal phosphorylation is due to unknown kinases rather than PKA. Login to comment 84 ABCC8 p.Ser1571Ala In addition, mutation of the PKA site (SUR1 S1571A) did not result in any significant decrease in phosphorylation in the basal state (Figure 4C, lane 7), and the PKA inhibitor H-89 did not reduce this basal phosphorylation (unpublished data), indicating that basal phosphorylation is due to unknown kinases rather than PKA. Login to comment 86 ABCC8 p.Ser1571Ala Digestion with trypsin of immunoprecipitated wild-type and the mutant SUR1 S1571A showed similar maps of the phosphopeptides. Login to comment 87 ABCC8 p.Ser1571Ala Digestion with trypsin of immunoprecipitated wild-type and the mutant SUR1 S1571A showed similar maps of the phosphopeptides. Login to comment 90 ABCC8 p.Ser1571Ala (A and B) Microsomal expression of wt Kir6.2/SUR1 and mutant Kir6.2 S372A/ SUR1 S1571A KATP channels in COS-1 cells. Login to comment 91 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala (A and B) Microsomal expression of wt Kir6.2/SUR1 and mutant Kir6.2 S372A/ SUR1 S1571A KATP channels in COS-1 cells. Login to comment 92 ABCC8 p.Ser1571Ala Untransfected control COS-1 cells (lane 1) or COS-1 cells transfected with wt Kir6.2 and wt SUR1 (lane 2) or mutant Kir6.2 S372A and SUR1 S1571A (lane 3) cDNA. Login to comment 98 ABCC8 p.Ser1571Ala To ascertain if a relatively high level of basal phosphorylation due to unknown kinases and/or PKA accounts for the failure to detect an increase in PKA-mediated SUR1 phosphorylation in intact cells, we eliminated possible unknown cytosolic kinases by preparing a crude membrane preparation from COS-1 cells transfected with the wt KATP channel (Kir6.2/SUR1) or the mutant KATP channel (Kir6.2 S372/SUR1 S1571A). Login to comment 99 ABCC8 p.Ser1571Ala To ascertain if a relatively high level of basal phosphorylation due to unknown kinases and/or PKA accounts for the failure to detect an increase in PKA-mediated SUR1 phosphorylation in intact cells, we eliminated possible unknown cytosolic kinases by preparing a crude membrane preparation from COS-1 cells transfected with the wt KATP channel (Kir6.2/SUR1) or the mutant KATP channel (Kir6.2 S372/SUR1 S1571A). Login to comment 102 ABCC8 p.Ser1571Ala Untransfected control COS-1 cells (lanes 1 and 2) or COS-1 cells transfected with wt Kir6.2 and wt SUR1 cDNAs (lanes 3 and 4) or the mutant Kir6.2 S372A and the mutant SUR1 S1571A cDNAs (lanes 5 and 6). Login to comment 103 ABCC8 p.Ser1571Ala Untransfected control COS-1 cells (lanes 1 and 2) or COS-1 cells transfected with wt Kir6.2 and wt SUR1 cDNAs (lanes 3 and 4) or the mutant Kir6.2 S372A and the mutant SUR1 S1571A cDNAs (lanes 5 and 6). Login to comment 109 ABCC8 p.Ser1571Ala Although a slight basal phosphorylation was still present, despite the expression levels of the wild-type and mutant SUR1 being similar (Figure 5A, insets 2 and 3), the addition of PKA substantially increased the phosphorylation of wt SUR1 and not that of mutant SUR1 S1571A (Figure 5B, compare lanes 3 and 4 with lanes 5 and 6, respectively). Login to comment 110 ABCC8 p.Ser1571Ala Although a slight basal phosphorylation was still present, despite the expression levels of the wild-type and mutant SUR1 being similar (Figure 5A, insets 2 and 3), the addition of PKA substantially increased the phosphorylation of wt SUR1 and not that of mutant SUR1 S1571A (Figure 5B, compare lanes 3 and 4 with lanes 5 and 6, respectively). Login to comment 112 ABCC8 p.Ser1571Ala For this purpose, COS-1 cells were transiently transfected with wild-type or the mutant (Kir6.2 S372A/SUR1 S1571A) KATP channel together with various Gs-coupled receptors. Login to comment 113 ABCC8 p.Ser1571Ala For this purpose, COS-1 cells were transiently transfected with wild-type or the mutant (Kir6.2 S372A/SUR1 S1571A) KATP channel together with various Gs-coupled receptors. Login to comment 121 ABCC8 p.Ser1571Ala COS-1 cells expressing endogenous b2;2-adrenergic receptor (lanes 9-12) or expressing various G-coupled receptors: PACAP receptor (lanes 1-4), GIP receptor (lanes 5-8) or somatostatin receptor (lanes 13-16) cDNA was co-transfected with wt Kir6.2 and wt SUR1 (lanes 1, 2, 5, 6, 9, 10, 13 and 14) or co-transfected with the mutant Kir6.2 S372A and SUR1 S1571A cDNAs (lanes 3, 4, 7, 8, 11, 12, 15 and 16). Login to comment 122 ABCC8 p.Ser1571Ala COS-1 cells expressing endogenous β2-adrenergic receptor (lanes 9-12) or expressing various G-coupled receptors: PACAP receptor (lanes 1-4), GIP receptor (lanes 5-8) or somatostatin receptor (lanes 13-16) cDNA was co-transfected with wt Kir6.2 and wt SUR1 (lanes 1, 2, 5, 6, 9, 10, 13 and 14) or co-transfected with the mutant Kir6.2 S372A and SUR1 S1571A cDNAs (lanes 3, 4, 7, 8, 11, 12, 15 and 16). Login to comment 140 ABCC8 p.Ser1571Ala As shown in Figure 7A and B, increase in activity was 2-fold for both wt Kir6.2/SUR1 (212%) and Kir6.2/SUR1 S1571A (209%). Login to comment 141 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala As shown in Figure 7A and B, increase in activity was 2-fold for both wt Kir6.2/SUR1 (212%) and Kir6.2/SUR1 S1571A (209%). Login to comment 142 ABCC8 p.Ser1571Ala In contrast, no significant increase in activity was observed in KATP channels comprising either Kir6.2 S372A/ wt SUR1 or Kir6.2 S372A/ SUR1 S1571A (Figure 7C and D). Login to comment 146 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala Current recordings were obtained in inside-out patches excised from COS-1 cells expressing wt Kir6.2 and wt SUR1 (A), wt Kir6.2 and the mutant SUR1 S1571A (B), the mutant Kir6.2 S372A and wt SUR1 (C), and the mutant Kir6.2 S372A and mutant SUR1 S1571A (D). Login to comment 147 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala Current recordings were obtained in inside-out patches excised from COS-1 cells expressing wt Kir6.2 and wt SUR1 (A), wt Kir6.2 and the mutant SUR1 S1571A (B), the mutant Kir6.2 S372A and wt SUR1 (C), and the mutant Kir6.2 S372A and mutant SUR1 S1571A (D). Login to comment 149 ABCC8 p.Ser1571Ala the KATP channels comprising wt SUR1 and those comprising the mutant SUR1 S1571A. Login to comment 150 ABCC8 p.Ser1571Ala the KATP channels comprising wt SUR1 and those comprising the mutant SUR1 S1571A. Login to comment 153 ABCC8 p.Ser1571Ala As shown in Figure 8 and Table I, the mean burst duration and the long closed time (c4;c3) of KATP channels comprising the mutant SUR1 S1571A were significantly (2-to 3-fold) and dramatically (6to 9-fold) prolonged, respectively, as compared with those of KATP channels comprising wt SUR1. Login to comment 154 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala As shown in Figure 8 and Table I, the mean burst duration and the long closed time (τc3) of KATP channels comprising the mutant SUR1 S1571A were significantly (2to 3-fold) and dramatically (6to 9-fold) prolonged, respectively, as compared with those of KATP channels comprising wt SUR1. Login to comment 155 ABCC8 p.Ser1571Ala The cluster duration in KATP channels comprising mutant SUR1 S1571A also was greatly prolonged (Figure 8, compare A or C with B or D; Table I) and, as a consequence, the open probability was increased (Table I). Login to comment 159 ABCC8 p.Ser1571Ala 4727 Treatment with alkaline phosphatase showed both PKA-dependent and -independent processes: a PKA-dependent process characterized by a transient increase in channel activity (1.7-fold) of the KATP channels comprising wt SUR1 (Figure 8A and C) that is abolished in the KATP channels comprising mutant SUR1 S1571A (Figure 8C and D) and a PKA-independent process observed as a decrease in channel activity of both wild-type and mutant KATP channels after long exposure to alkaline phosphatase (Figure 8, right). Login to comment 160 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala 4727 Treatment with alkaline phosphatase showed both PKA-dependent and -independent processes: a PKA-dependent process characterized by a transient increase in channel activity (1.7-fold) of the KATP channels comprising wt SUR1 (Figure 8A and C) that is abolished in the KATP channels comprising mutant SUR1 S1571A (Figure 8C and D) and a PKA-independent process observed as a decrease in channel activity of both wild-type and mutant KATP channels after long exposure to alkaline phosphatase (Figure 8, right). Login to comment 161 ABCC8 p.Ser1571Ala Treatment with alkaline phosphatase also prolonged significantly burst and cluster duration, and to some extent the long closed time (τc3) of the KATP channels comprising wt SUR1 (Figure 8A and C; Table II), while no significant change was observed in the KATP channels comprising mutant SUR1 S1571A (Figure 8B and D; Table II). Login to comment 163 ABCC8 p.Ser1571Ala Although alkaline phosphatase cleaves any of the phosphate groups that are phosphorylated by PKA and unknown kinases, the insensitivity of mutant SUR1 S1571A to alkaline phosphatase after short exposure indicates that the burst and cluster duration, interburst interval and open probability effects are due to the PKA rather than unknown kinase phosphorylation. Login to comment 164 ABCC8 p.Ser1571Ala Although alkaline phosphatase cleaves any of the phosphate groups that are phosphorylated by PKA and unknown kinases, the insensitivity of mutant SUR1 S1571A to alkaline phosphatase after short exposure indicates that the burst and cluster duration, interburst interval and open probability effects are due to the PKA rather than unknown kinase phosphorylation. Login to comment 168 ABCC8 p.Ser1571Asp ABCC8 p.Ser1571Asp In contrast, no significant change was observed in open probability (0.12 afe; 0.06 and 0.09 afe; 0.03, for wt Kir6.2/SUR1 and wt Kir6.2/ SUR1 S1571D, respectively, n afd; 4-7) or channel kinetics (unpublished data) for the SUR1 S1571D mutant, as would be expected if SUR1 is already phosphorylated under basal conditions. Login to comment 169 ABCC8 p.Ser1571Asp ABCC8 p.Ser1571Asp In contrast, no significant change was observed in open probability (0.12 Ϯ 0.06 and 0.09 Ϯ 0.03, for wt Kir6.2/SUR1 and wt Kir6.2/ SUR1 S1571D, respectively, n ϭ 4-7) or channel kinetics (unpublished data) for the SUR1 S1571D mutant, as would be expected if SUR1 is already phosphorylated under basal conditions. Login to comment 170 ABCC8 p.Ser1571Ala Interestingly, rubidium efflux from the KATP channels comprising SUR1 S1571A expressed in COS-1 cells decreased by 68 afe; 12% (n afd; 3), compared with that from wt KATP channels. Login to comment 171 ABCC8 p.Ser1571Ala Interestingly, rubidium efflux from the KATP channels comprising SUR1 S1571A expressed in COS-1 cells decreased by 68 Ϯ 12% (n ϭ 3), compared with that from wt KATP channels. Login to comment 174 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala Single channels were obtained in inside-out patches excised from COS-1 cells expressing wt Kir6.2 and wt SUR1 (A), wt Kir6.2 and the mutant SUR1 S1571A (B), the mutant Kir6.2 S372A and wt SUR1 (C), the mutant Kir6.2 S372A and mutant SUR1 S1571A (D). Login to comment 175 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala Single channels were obtained in inside-out patches excised from COS-1 cells expressing wt Kir6.2 and wt SUR1 (A), wt Kir6.2 and the mutant SUR1 S1571A (B), the mutant Kir6.2 S372A and wt SUR1 (C), the mutant Kir6.2 S372A and mutant SUR1 S1571A (D). Login to comment 176 ABCC8 p.Ser1571Ala were markedly reduced in cells transfected with mutant SUR1 S1571A (Table I). Login to comment 177 ABCC8 p.Ser1571Ala were markedly reduced in cells transfected with mutant SUR1 S1571A (Table I). Login to comment 178 ABCC8 p.Ser1571Ala Using channel density and open probability parameters, the channel activity of KATP channel comprising SUR1 S1571A can be estimated as 30-40% of wt KATP channels. Login to comment 179 ABCC8 p.Ser1571Ala Using channel density and open probability parameters, the channel activity of KATP channel comprising SUR1 S1571A can be estimated as 30-40% of wt KATP channels. Login to comment 188 ABCC8 p.Ser1571Ala However, the simple replacement of the PKA phosphorylation site at serine-1571 with alanine in human SUR1 prolonged the burst and cluster duration, interburst interval (c4;c3) and increased the open probability. Login to comment 189 ABCC8 p.Ser1571Ala However, the simple replacement of the PKA phosphorylation site at serine-1571 with alanine in human SUR1 prolonged the burst and cluster duration, interburst interval (τc3) and increased the open probability. Login to comment 190 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala Properties of wild-type and various mutant K ATP channels K ATP channel Open probability Burst duration Cluster duration Intraburst kinetics (ms) Interburst kinetics (ms) Channel density (Po) (ms) (ms) Open time (c4; o ) Short closed Long closed time Detectable rate Channel number time (c4; c1 ) (c4; c2 ) (c4; c3 ) (%) in a patch Kir6.2 wt/SUR1 wt 0.12 afe; 0.06 25 afe; 11 75 afe; 18 1.94 afe; 0.19 0.49 afe; 0.03 10.1 afe; 2.9 131 afe; 66 66.3 26.4 afe; 6.5 Kir6.2 wt/SUR1 S1571A 0.44 afe; 0.02 a 73 afe; 17 a 394 afe; 113 a 2.21 afe; 0.13 0.44 afe; 0.03 12.9 afe; 4.5 1120 afe; 71 a 40.0 4.8 afe; 0.8 a Kir6.2 S372A/SUR1 wt 0.13 afe; 0.03 35 afe; 12 75 afe; 12 1.92 afe; 0.10 0.49 afe; 0.02 8.0 afe; 2.6 102 afe; 36 65.0 19.2 afe; 3.4 Kir6.2 S372A/SUR1 S1571A 0.62 afe; 0.08 a 74 afe; 20 a 274 afe; 37 a 2.22 afe; 0.30 0.49 afe; 0.04 10.5 afe; 1.0 863 afe; 49 a 35.0 3.2 afe; 0.4 a Recordings were made at -70 mV in ATP-free conditions [n afd; 5-7 in all cases, except for the channel density determination (n afd; 60-80)]. Login to comment 201 ABCC8 p.Ser1571Asp Since the PKA action of each subunit can be mimicked by the Kir6.2 (S372D) or SUR1 (S1571D) mutant, the negative charges carried by both subunits seem to be good candidates for this structural change. Login to comment 208 ABCC8 p.Ser1571Ala On the other hand, since functional expression of the channels at the cell surface is reduced in SUR1 S1571A, the PKA phosphorylation in SUR1 might also be involved in the trafficking from the intracellular vesicles Table II. Login to comment 209 ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala ABCC8 p.Ser1571Ala Properties of wild-type and various mutant KATP channels after AP treatment KATP channel Burst duration (ms) Cluster duration (ms) Long closed time (τc3) Before alkaline phosphatase Kir6.2 wt/SUR1 wt 15.7 Ϯ 2.4 40 Ϯ 13 186 Ϯ 53 Kir6.2 wt/SUR1 S1571A 26.6 Ϯ 7.6 152 Ϯ 35 1318 Ϯ 208 Kir6.2 S372A/SUR1 wt 18.7 Ϯ 3.0 59 Ϯ 5 197 Ϯ 57 Kir6.2 S372A/SUR1 S1571A 53.6 Ϯ 3.7 169 Ϯ 19 1654 Ϯ 594 After alkaline phosphatase Kir6.2 wt/SUR1 wt 50.3 Ϯ 9.2a 155 Ϯ 32a 402 Ϯ 60 Kir6.2 wt/SUR1 S1571A 36.9 Ϯ 11.1 169 Ϯ 41 1236 Ϯ 279 Kir6.2 S372A/SUR1 wt 41.1 Ϯ 3.2a 103 Ϯ 15a 576 Ϯ 162 Kir6.2 S372A/SUR1 S1571A 52.6 Ϯ 6.1 178 Ϯ 37 1743 Ϯ 485 Recordings were made at -70 mV in 10 µM ATP conditions (n ϭ 3-6 in all cases). Login to comment