ABCMdb

PMID: 9238070

Trapp S, Tucker SJ, Ashcroft FM
Activation and inhibition of K-ATP currents by guanine nucleotides is mediated by different channel subunits.
Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8872-7., [PubMed]

Sentences

No. Mutations Sentence Comment

14 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met We also showed that mutation of the lysine residue in the Walker A motif of either the first (K719A) or second (K1384M) nucleotide-binding domain of SUR1 abolished both the potentiatory effects of GTP and GDP on K-ATP currents and their ability to support stimulation by diazoxide. Login to comment 38 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met We examined the effects of mutating the WA lysine in either the first (K719A) or second (K1384M) NBD. Login to comment 95 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met We refer to these as K719A (wild-type Kir6.2 coexpressed with K719A-SUR1) and K1384M (wild-type Kir6.2 coexpressed with K1384M-SUR1). Login to comment 96 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met In contrast to wild-type K-ATP currents, neither K719A nor K1384M currents were activated by GTP (Fig. 2A). Login to comment 97 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Instead, increasing GTP concentrations simply produced a dose-dependent inhibition of both K719A and K1384M currents (Fig. 2B). Login to comment 98 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met For K719A currents, the Ki for current inhibition was 2.7 Ϯ 0.6 mM and the Hill coefficient was 1.0 Ϯ 0.2 (n ϭ 5), and for K1384M currents, the Ki was 3.3 Ϯ 0.5 mM and the Hill coefficient was 1.4 Ϯ 0.2 (n ϭ 5). Login to comment 102 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met (A) Macroscopic currents recorded from three different inside-out patches in response to a series of voltage ramps from -110 to ϩ100 mV. Oocytes were coinjected with mRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, or K1384M-SUR1 mRNAs. Login to comment 111 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Neither K719A nor K1384M currents were activated by 100 ␮M GDP. Login to comment 115 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The lack of an inhibitory effect of 1 mM GDP on K719A or K1384M currents suggests that at this concentration GDP exerts only a stimulatory effect on wild-type K-ATP currents. Login to comment 116 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met MgGDP (10 mM) blocked K719A and K1384M currents by 30 Ϯ 5% and by 55 Ϯ 6%, respectively (Fig. 3C). Login to comment 117 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The extent of this inhibition suggests that GDP does not interact as strongly as ADP with the inhibitory binding site, since as little as 100 ␮M ADP blocked K719A and K1384M currents by Ͼ60% (13). Login to comment 121 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met We therefore examined the ability of guanine nucleotides to support the action of diazoxide on wild-type, K719A, and K1384M currents. Login to comment 131 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met (A) Macroscopic currents recorded from three different inside-out patches in response to a series of voltage ramps from -110 to ϩ100 mV. Oocytes were coinjected with mRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, or K1384M-SUR1. Login to comment 136 ABCC8 p.Lys719Ala The broken line indicates the control (GDP-free) conductance level. The number of patches is indicated above the bars. Diazoxide produced a slight (but significant, P Ͻ 0.05) block of K719A currents when either GTP or GDP was present. Login to comment 138 ABCC8 p.Lys1384Met Diazoxide was without effect on K1384M currents in the presence of guanine nucleotides. Login to comment 144 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The efficacy of block was comparable to that observed for wild-type Kir6.2 when coexpressed with mutant SUR1: half-maximal inhibition was 6.0 mM for Kir6.2⌬C26 currents, 2.7 mM for K719A currents, and 3.3 mM for K1384M currents. Login to comment 148 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met This may explain why the K719A and K1384M currents appear slightly more sensitive to GTP than Kir6.2⌬C26 currents. Login to comment 155 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met (A) Macroscopic currents recorded from three different inside-out patches in response to a series of voltage ramps from -110 to ϩ100 mV. Oocytes were coinjected with mRNAs encoding Kir6.2 and either wild-type SUR1, K719A-SUR1, or K1384M-SUR1. Login to comment 160 ABCC8 p.Lys719Ala Inhibition of K719A currents by diazoxide was significant both in the presence of GTP (P Ͻ 0.05) or GDP (P Ͻ 0.005). Login to comment 167 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met One explanation for the inability of guanine nucleotides to enhance K719A or K1384M currents is that mutation of the WA lysines abolishes binding of either GTP or GDP. Login to comment 172 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The fact that neither GTP nor GDP activate native K-ATP currents in the absence on Mg2ϩ (13), or K719A or K1384M currents in the presence of Mg2ϩ , is consistent with this hypothesis. Login to comment 174 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met We further show that mutation of the WA lysine at NBD1 (K719A) completely abolishes, while mutation of that at NBD2 (K1384M) very substantially reduces, the ability of GTP or GDP to support diazoxide activation. Login to comment