ABCMdb

PMID: 9350615

Gribble FM, Tucker SJ, Ashcroft FM
The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation.
J Physiol. 1997 Oct 1;504 ( Pt 1):35-45., [PubMed]

Sentences

No. Mutations Sentence Comment

13 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met When SUR1 was mutated to abolish MgADP activation of KATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. Login to comment 15 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The Ki for tolbutamide interaction with either the high- or low-affinity site was unaffected by 100 /SM MgADP, for both wild-type and K719A-K1384M currents. Login to comment 17 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met MgGDP (100 /M) enhanced wild-type KATP currents and was without effect on K719A-K1384M currents. Login to comment 55 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The lysine residues at position 719 or 1384 of SUR1 were replaced by an alanine or methionine, respectively (K719A, K1384M). Login to comment 58 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala All SUR1 mutations were coexpressed with wild-type Kir6.2 and the resulting currents are referred to by the SUR1 mutation only (thus, wild-type Kir6.2 plus SUR1-K719A currents are referred to as K719A currents). Login to comment 107 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met Mutation of the WA lysine residues in either NBD1 (K719A) or NBD2 (K1384M), or both (K719A-K1384M) prevented the MgADP activation of KATP currents and unmasked the inhibitory effect of MgADP (Figs 2C and 4; Gribble et al. 1997 b). Login to comment 108 ABCC8 p.Lys719Ala ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met ABCC8 p.Lys1384Met In the absence A 1 nA Tolbutamide B 30 s 0-5 nA MgADP Tolbutamide of nucleotides, the extent of block of K719A-K1384M, K719A and K1384M currents by 0 5 mm tolbutamide was similar to that of wild-type channels (Fig. 3). Login to comment 111 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Effects of MgADP and tolbutamide on K719A-K1384M currents Macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV (holding potential, 0 mV). Login to comment 112 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The oocytes were coinjected with mRNAs encoding wild-type Kir6.2 and K719A-K1384M SUR1. Login to comment 122 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met K719A-K1384M currents by 67 % (Fig. 2C). Login to comment 124 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Furthermore, MgADP did not enhance tolbutamide inhibition of K719A-K1384M currents (Figs 2 and 3). Login to comment 125 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Similar results were observed for K719A and K1384M currents (Figs 3 and 4), which confirms that the WA lysine at each of the NBDs of SUR1 is needed both for the stimulatory effect of MgADP itself 150 - 100 -1. Login to comment 127 ABCC8 p.Asp853Asn ABCC8 p.Asp1505Asn Mutation of the WB aspartate residues in either NBD1 (D853N) or NBD2 (D1505N) of SURI abolished the stimulatory effects of MgADP on KATP currents (Fig. 4) and prevented the MgADP-dependent enhancement of tolbutamide block (Fig. 3). Login to comment 141 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met O (dashed line), K719A-K1384M currents (n = 6). Login to comment 144 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met K719A-K1384M channel currents: K11 = 1'6 /M, h1 = 0-7, K42 = 2-1 mM, h2==14, L = 0'40. Login to comment 152 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met O, K719A-K1384M currents, 100 /uM MgADP (n = 6). Login to comment 154 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met In the presence of 100 ,UM MgADP: Kj1 = 6-6 ,UM, h1 = 1.1, K12= 43 mM, h2= 1-0, L = 0-13,A =4-3, B= 0-33, for wild-type channel currents; and Kj1= 3-1 um, h1= 12, K12=5-5mM, h2=1,L=044,A=1 0,B=033,for K719A-K1384M currents. Login to comment 158 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met A, K719A-K1384M currents, 100 /uM MgGDP (n = 6). Login to comment 160 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met In the presence of 100 /uM MgGDP: Kj1 = 60 FuM, h1 = 1 0, K12 = 2-0 mm, h2 = 1 0, L = 0.39, A = 1 9, B = 0 9, for wild-type channel currents; and Ki = 3-8 FuM, h,= 10, K2= 28 mM, h2= 1-3, L= 0-56, A = 1 0, B= 0-9, for K719A-K1384M currents. Login to comment 161 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met 1000 100000.01 0.1 1 10 100 [Tolbutamide] (uM) 40 J. Physiol.504.1 We next examined the dose-response curve for tolbutamide block of K719A-K1384M currents. Login to comment 172 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Unlike wild-type KATP currents, K719A-K1384M channel currents were blocked by 100 /LM MgADP (by 63-4 + 2-8%, n = 17). Login to comment 175 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met The best fit to the mutant channel currents in 100 /M MgADP was obtained with a K1 of 3-1 uM (2-1-4-7 /tM, n = 5), which is not significantly different (ANOVA) from that of wild-type currents, or K719A-K1384M currents in the absence of ADP. Login to comment 178 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met This would account for the fact that MgADP has no effect on the tolbutamide block of K719A-K1384M currents, which are not stimulated by the nucleotide. Login to comment 189 ABCC8 p.Lys719Ala In contrast to MgADP, 100 ,UM MgGDP had little effect on K719A-K1 384M currents, which were 90 + 3% (n = 6) of their amplitude in control solution. Login to comment 192 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met This confirms that MgGDP is able to act at the stimulatory nucleotide-binding site in wild-type, but not K719A-K1384M, channels. Login to comment 237 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Second, the tolbutamide block of K719A-K1384M currents, which are not potentiated by MgADP, was not altered by MgADP. Login to comment 240 ABCC8 p.Lys719Ala ABCC8 p.Lys1384Met Since Mg2+ is required for the stimulatory effects of MgADP and MgGDP (Bokvist et al. 1991; Gribble et al. 1997 b), our results may also explain why nucleotide diphosphates only influence tolbutamide inhibition of KATP currents in the presence of Mg2+; in the absence of Mg2+, the effects of ADP would resemble those found for K719A-K1384M currents. Login to comment