ABCMdb

PMID: 16023110

Yang K, Fang K, Fromondi L, Chan KW
Low temperature completely rescues the function of two misfolded K ATP channel disease-mutants.
FEBS Lett. 2005 Aug 1;579(19):4113-8., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Two SUR1 mutations, A116P and V187D, reduce channel activity causing persistent hyperinsulinemic hypoglycemia of infancy. Login to comment 17 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Two SUR1 mutations, A116P and V187D, have been reported to cause PHHI (Fig. 1) [7,8]. Login to comment 60 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Maturation of A116P and V187D SUR1 is temperature sensitive and requires Kir6.2 We tested whether the maturation of A116P and V187D SUR1 was temperature sensitive by co-expressing them with Kir6.2 at three different temperatures. Login to comment 65 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Only the lower band was found for either A116P or V187D, indicating that they were retained in the ER. Login to comment 67 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro At 30 °C, both mutants could form the mature upper band but the percentage of the upper band normalized to that of the WT was only 33% for A116P and 86% for V187D (Fig. 2A and B). Login to comment 68 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Interestingly, at 18 °C, similar proportions of WT and mutants matured to form the upper bands (relative to WT, 96% for A116P and 99% for V187D). Login to comment 70 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The reduced rescue in the processing of A116P compared to V187D at Fig. 1. Login to comment 73 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro SUR1 is made up of TMD0 and the core domain connected together through the cytoplasmic linker L0. A116P and V187D are two mutations that cause PHHI. Login to comment 76 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Processing and misfolding of A116P and V187D mutants are temperature sensitive. Login to comment 81 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, the upper band was not detected for A116P-AAA and V187D-AAA. Login to comment 82 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro However, the upper band was not detected for A116P-AAA and V187D-AAA. Login to comment 85 ABCC8 p.Ala116Pro 30 °C indicates that A116P causes more severe perturbation in the processing of SUR1. Login to comment 86 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro When expressed at 37 °C in the absence of Kir6.2, WT SUR1 but neither A116P nor V187D have been shown to be mature glycosylated when their RKR motifs were mutated to AAA (three alanines) [9]. Login to comment 87 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro We found that even at 18 °C, A116P and V187D SUR1-AAA could not mature to form the upper band (Fig. 2B) in the absence of Kir6.2. Login to comment 88 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro When expressed at 37 C in the absence of Kir6.2, WT SUR1 but neither A116P nor V187D have been shown to be mature glycosylated when their RKR motifs were mutated to AAA (three alanines) [9]. Login to comment 89 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We found that even at 18 C, A116P and V187D SUR1-AAA could not mature to form the upper band (Fig. 2B) in the absence of Kir6.2. Login to comment 90 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro A116P and V187D disrupt heteromeric subunits interaction at 37 °C, but not at 18 °C, by causing misfolding One explanation for the temperature sensitive processing of these mutants is that the mutations cause misfolding in SUR1 that is temperature sensitive. Login to comment 92 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro A116P and V187D disrupt heteromeric subunits interaction at 37 C, but not at 18 C, by causing misfolding One explanation for the temperature sensitive processing of these mutants is that the mutations cause misfolding in SUR1 that is temperature sensitive. Login to comment 102 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro We have previously shown that A116P or V187D mutation abolished the association between TMD0 and Kir6.2 at 18 °C [5], which seems to contradict with our current result obtained at the same temperature. Login to comment 106 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Mutant and the WT channels show indistinguishable channel properties at 18 °C suggesting folding defect is completely corrected It is possible that A116P and V187D mutations still cause small conformational changes in SUR1 that are not detected by immunoprecipitation when expressed at 18 °C. Login to comment 110 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Mutant and the WT channels show indistinguishable channel properties at 18 C suggesting folding defect is completely corrected It is possible that A116P and V187D mutations still cause small conformational changes in SUR1 that are not detected by immunoprecipitation when expressed at 18 C. Login to comment 115 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro t1/2 for azide activation is the time required to reach half of the maximally azide activated current (t1/2 for WT = 264 ± 10 s; A116P = 246 ± 10 s; and V187D = 277 ± 10 s; n = 6). Login to comment 116 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Azide activated current is the maximum current obtained in the presence of azide minus the background current remained in the presence of glibenclamide (Iaz for WT = À14.53 ± 2.66 lA; A116P = À15.96 ± 3.98 lA; V187D = À14.18 ± 2.80 lA). Login to comment 117 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Diazoxide/azide activation is the ratio of the current activated by diazoxide divided by the current activated by azide (Idzx/Iaz for WT = 0.95 ± 0.08; A116P = 0.92 ± 0.13; V187D = 1.12 ± 0.10). Login to comment 119 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro t1/2 for azide activation is the time required to reach half of the maximally azide activated current (t1/2 for WT = 264 &#b1; 10 s; A116P = 246 &#b1; 10 s; and V187D = 277 &#b1; 10 s; n = 6). Login to comment 120 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Azide activated current is the maximum current obtained in the presence of azide minus the background current remained in the presence of glibenclamide (Iaz for WT = 14.53 &#b1; 2.66 lA; A116P = 15.96 &#b1; 3.98 lA; V187D = 14.18 &#b1; 2.80 lA). Login to comment 121 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Diazoxide/azide activation is the ratio of the current activated by diazoxide divided by the current activated by azide (Idzx/Iaz for WT = 0.95 &#b1; 0.08; A116P = 0.92 &#b1; 0.13; V187D = 1.12 &#b1; 0.10). Login to comment 127 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Lastly, we investigated the effect of A116P and V187D mutations on the single channel characteristics of the SUR1/Kir6.2 channels. Login to comment 131 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Lastly, we investigated the effect of A116P and V187D mutations on the single channel characteristics of the SUR1/Kir6.2 channels. Login to comment 134 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro (A) Representative current traces obtained at À100 mV from patches excised from oocytes expressing WT or A116P SUR1/Kir6.2 channels (trace for V187D mutant channels were not shown). Login to comment 139 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro The obtained values for k (lM) and n are: 16.71 ± 0.88 (k) and 1.43 ± 0.09 (n) for WT; 12.17 ± 0.25 and 1.17 ± 0.03 for A116P; 11.21 ± 0.22 and 1.42 ± 0.04 for V187D. Login to comment 144 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro The obtained values for k (lM) and n are: 16.71 &#b1; 0.88 (k) and 1.43 &#b1; 0.09 (n) for WT; 12.17 &#b1; 0.25 and 1.17 &#b1; 0.03 for A116P; 11.21 &#b1; 0.22 and 1.42 &#b1; 0.04 for V187D. Login to comment 146 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Table 1 Single channel parameters for WT, A116P and V187D SUR1/Kir6.2 channelsa SUR1 + Kir6.2 A116P + Kir6.2 V187D + Kir6.2 sc1 (ms) 0.36 ± 0.002 0.43 ± 0.007 0.38 ± 0.007 sc2 (ms) 9.36 ± 2.238 7.28 ± 1.711 10.37 ± 2.895 sc3 (ms) 63.12 ± 14.543 42.92 ± 7.702 71.31 ± 37.949 ac1 0.969 ± 0.006 0.961 ± 0.008 0.975 ± 0.0006 ac2 0.025 ± 0.007 0.025 ± 0.005 0.016 ± 0.003 ac3 0.006 ± 0.002 0.014 ± 0.003 0.009 ± 0.003 so (ms) 1.31 ± 0.042 1.47 ± 0.027 1.46 ± 0.056 sb (ms) 67.66 ± 11.93 68.92 ± 17.77 77.96 ± 4.06 Po 0.595 ± 0.034 0.554 ± 0.047 0.583 ± 0.022 i (pA) 6.05 ± 0.43 6.07 ± 0.06 6.26 ± 0.17 a Explanations of the symbols can be found in the legends of Figs. 5 and 6. Login to comment 148 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Conclusion Our data prove that A116P and V187D disrupt the association between the two KATP channel subunits by causing misfolding in SUR1 at physiological temperature (37 °C). Login to comment 151 ABCC8 p.Val187Asp ABCC8 p.Val187Asp ABCC8 p.Ala116Pro ABCC8 p.Ala116Pro Table 1 Single channel parameters for WT, A116P and V187D SUR1/Kir6.2 channelsa SUR1 + Kir6.2 A116P + Kir6.2 V187D + Kir6.2 sc1 (ms) 0.36 &#b1; 0.002 0.43 &#b1; 0.007 0.38 &#b1; 0.007 sc2 (ms) 9.36 &#b1; 2.238 7.28 &#b1; 1.711 10.37 &#b1; 2.895 sc3 (ms) 63.12 &#b1; 14.543 42.92 &#b1; 7.702 71.31 &#b1; 37.949 ac1 0.969 &#b1; 0.006 0.961 &#b1; 0.008 0.975 &#b1; 0.0006 ac2 0.025 &#b1; 0.007 0.025 &#b1; 0.005 0.016 &#b1; 0.003 ac3 0.006 &#b1; 0.002 0.014 &#b1; 0.003 0.009 &#b1; 0.003 so (ms) 1.31 &#b1; 0.042 1.47 &#b1; 0.027 1.46 &#b1; 0.056 sb (ms) 67.66 &#b1; 11.93 68.92 &#b1; 17.77 77.96 &#b1; 4.06 Po 0.595 &#b1; 0.034 0.554 &#b1; 0.047 0.583 &#b1; 0.022 i (pA) 6.05 &#b1; 0.43 6.07 &#b1; 0.06 6.26 &#b1; 0.17 a Explanations of the symbols can be found in the legends of Figs. 5 and 6. Login to comment 153 ABCC8 p.Val187Asp ABCC8 p.Ala116Pro Conclusion Our data prove that A116P and V187D disrupt the association between the two KATP channel subunits by causing misfolding in SUR1 at physiological temperature (37 C). Login to comment