ABCMdb

PMID: 19139106

Winkler M, Lutz R, Russ U, Quast U, Bryan J
Analysis of two KCNJ11 neonatal diabetes mutations, V59G and V59A, and the analogous KCNJ8 I60G substitution: differences between the channel subtypes formed with SUR1.
J Biol Chem. 2009 Mar 13;284(11):6752-62. Epub 2009 Jan 12., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC8 p.Ile60Gly We compared Kir6.x/SUR1 channels carrying the V59G substitution, a cause of the developmental delay, epilepsy, and neonatal diabetes syndrome, with a V59A substitution and the equivalent I60G mutation in the related Kir6.1 subunit from vascular smooth muscle. Login to comment 10 ABCC8 p.Ile60Gly The I60G channels, strongly dependent on SUR stimulation, remain sensitive to sulfonylureas. Login to comment 50 ABCC8 p.Ile60Gly To support this hypothesis, we sought to characterize the V59G mutation further, compare it with a V59A substitution and the analogous substitution, I60G, in Kir6.1 the pore-forming subunit of the vascular KATP channel. Login to comment 51 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly This channel is composed of Kir6.1 and SUR2B (40), however, for comparison we examined the I60G/SUR1 channel. Login to comment 52 ABCC8 p.Ile60Gly This channel is composed of Kir6.1 and SUR2B (40), however, for comparison we examined the I60G/SUR1 channel. Login to comment 56 ABCC8 p.Ile60Gly In view of these differences in gating on one hand and of the similarity in amino acid sequence around the mutation on the other it was of interest to compare the V59G and I60G/ SUR1 KATP channels. Login to comment 57 ABCC8 p.Ile60Gly In view of these differences in gating on one hand and of the similarity in amino acid sequence around the mutation on the other it was of interest to compare the V59G and I60G/ SUR1 KATP channels. Login to comment 60 ABCC8 p.Ile60Gly The V59G, V59A, and I60G mutations were introduced into Kir6.2 and Kir6.1, respectively, using the QuikChange II XL site-directed mutagenesis kit (Stratagene). Login to comment 61 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly The mutations were verified by sequencing the relevant DNA region (V59G) or the whole coding region (I60G and V59A). Login to comment 62 ABCC8 p.Ile60Gly The mutations were verified by sequencing the relevant DNA region (V59G) or the whole coding region (I60G and V59A). Login to comment 96 ABCC8 p.Ile60Gly The same structural predictions were obtained for the I60G substitution in Kir6.1. Login to comment 97 ABCC8 p.Ile60Gly The same structural predictions were obtained for the I60G substitution in Kir6.1. Login to comment 173 ABCC8 p.Ile60Gly Response of Kir6.1/SUR1 Channels to Nucleotides and Sulfonylureas-Fig. 6 compares the responses of cells expressing wild-type versus I60G/SUR1 channels to stimulation with 1 mM MgATP/0.3 mM MgGDP. Login to comment 174 ABCC8 p.Ile60Gly Response of Kir6.1/SUR1 Channels to Nucleotides and Sulfonylureas-Fig. 6 compares the responses of cells expressing wild-type versus I60G/SUR1 channels to stimulation with 1 mM MgATP/0.3 mM MgGDP. Login to comment 175 ABCC8 p.Ile60Gly An immediate, stable current was also obtained when cells expressing the I60G channels were dialyzed with 10 mM MgATP indicating that 10 mM MgATP did not close the mutant channel (n ϭ 5). Login to comment 176 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly Dialysis with Mg2ϩ - or nucleotide-free solutions led to a steady loss of current over 2-10 min indicating that the I60G channel required activation to remain in the open state (n ϭ 12). Login to comment 177 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly GBC (10 nM) inhibited both the wild-type and I60G channels Ն90% (Fig. 6). Login to comment 178 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly This inhibition of the I60G channels is in sharp contrast to the V59G Kir6.2 channels (Fig. 1b), which were insensitive to GBC up to 1 ␮M under equivalent activating conditions. Login to comment 179 ABCC8 p.Ile60Gly This inhibition of the I60G channels is in sharp contrast to the V59G Kir6.2 channels (Fig. 1b), which were insensitive to GBC up to 1 òe;M under equivalent activating conditions. Login to comment 181 ABCC8 p.Ile60Gly In the whole cell configuration, wild-type Kir6.1/SUR1 channels were completely and reversibly inhibited by tolbutamide (100 ␮M), whereas the I60G channels were 89 Ϯ 6 and 91 Ϯ 4% inhibited by 100 and 300 ␮M tolbutamide (n ϭ 4 and 8), respectively. Login to comment 182 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly Assuming complete inhibition, an IC50 value of ϳ10 ␮M was estimated for the I60G channel. Login to comment 183 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly The experiments in the whole cell configuration (Fig. 6) suggest that, in the cellular environment, wild-type Kir6.1 channels are essentially closed, whereas the I60G channels are open. Login to comment 184 ABCC8 p.Ile60Gly The experiments in the whole cell configuration (Fig. 6) suggest that, in the cellular environment, wild-type Kir6.1 channels are essentially closed, whereas the I60G channels are open. Login to comment 188 ABCC8 p.Ile60Gly As shown in Fig. 7b, the I60G channels were spontaneously active in on-cell patches and exhibited transitions between long lived states that differed by ϳ1.8 pA. Login to comment 189 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly Tolbutamide (300 ␮M) reversibly inhibited the I60G channels (Fig. 7b), whereas diazoxide had no effect (n ϭ 9). Login to comment 190 ABCC8 p.Ile60Gly Tolbutamide (300 òe;M) reversibly inhibited the I60G channels (Fig. 7b), whereas diazoxide had no effect (n afd; 9). Login to comment 191 ABCC8 p.Ile60Gly DISCUSSION Structural Hypothesis-We compared Val-59-substituted Kir6.2/SUR1 and analogous I60G Kir6.1/SUR1 channels with their respective wild-type channels. Login to comment 192 ABCC8 p.Ile60Gly DISCUSSION Structural Hypothesis-We compared Val-59-substituted Kir6.2/SUR1 and analogous I60G Kir6.1/SUR1 channels with their respective wild-type channels. Login to comment 233 ABCC8 p.Ile60Gly Whole cell and cell-attached recordings show that the I60G channels are more active than wild-type in intact cells (Fig. 7). Login to comment 234 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly Whole cell and cell-attached recordings show that the I60G channels are more active than wild-type in intact cells (Fig. 7). Login to comment 235 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly This suggests that under physiologic conditions the I60G channels either have a reduced sensitivity to inhibitory ATP versus wild-type or are more efficiently activated by MgATP. Login to comment 236 ABCC8 p.Ile60Gly We did not observe I60G channel activity in the absence of Mg2ϩ and activating nucleotides consistent with earlier studies on the Kir6.1/SUR1 wild-type (34, 40, 42, 43), whereas both the Val-59-substituted and wild-type Kir6.2 channels are open in the absence of activating nucleotides (e.g. Figs. Login to comment 267 ABCC8 p.Ile60Gly Second, the I60G channels, whose activity is strongly dependent on Mg-nucleotide-dependent stimulation by SUR1, are nearly as sensitive to GBC as the wild-type (potency difference b03; 4afb;). Login to comment 268 ABCC8 p.Ile60Gly ABCC8 p.Ile60Gly Second, the I60G channels, whose activity is strongly dependent on Mg-nucleotide-dependent stimulation by SUR1, are nearly as sensitive to GBC as the wild-type (potency difference ϳ 4ϫ). Login to comment 269 ABCC8 p.Ile60Gly GBC limits the stimulatory input from SUR1 thereby inducing closure of the I60G channels. Login to comment