ABCMdb

PMID: 21321069

Pratt EB, Tewson P, Bruederle CE, Skach WR, Shyng SL
N-terminal transmembrane domain of SUR1 controls gating of Kir6.2 by modulating channel sensitivity to PIP2.
J Gen Physiol. 2011 Mar;137(3):299-314. Epub 2011 Feb 14., [PubMed]

Sentences

No. Mutations Sentence Comment

17 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Using two previously described disease-causing mutations in TMD0 (R74W and E128K), we performed amino acid substitutions to study the structural roles of these residues in KATP channel function in the context of full-length SUR1 as well as TMD0. Login to comment 18 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Our results revealed that although R74W and E128K in full-length SUR1 both decrease surface channel expression and reduce channel sensitivity to ATP inhibition, they arrive there via distinct mechanisms. Login to comment 21 ABCC8 p.Glu128Lys Importantly, E128K full-length channels, despite having a greatly reduced Po, exhibit little response to phosphatidylinositol 4,5-bisphosphate (PIP2) stimulation. Login to comment 23 ABCC8 p.Glu128Trp Further supporting this notion, the E128W mutation in full-length channels resulted in channel inactivation that was prevented or reversed by exogenous PIP2. Login to comment 30 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Recently, we reported that two mutations (R74W and E128K) in the TMD0 domain of SUR1 identified in congenital hyperinsulinism cause loss of channel function by preventing channel trafficking to the cell surface. Login to comment 32 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp The R74W- and E128K-mutant channels exhibit reduced channel sensitivity to ATP inhibition. Login to comment 33 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp However, unlike most ATP-insensitive mutants in which an increased Po underlies the reduction in apparent ATP sensitivity by allosteric effects, the R74W and E128K mutants display decreased Po (Pratt et al., 2009). Login to comment 35 ABCC8 p.Arg74* ABCC8 p.Glu128* In this study, we systematically replaced residues 74 and 128 with other amino acids (referred to as R74X and E128X) in full-length and mini-KATP channels to probe their structural and functional roles in the coupling of TMD0 to Kir6.2. Login to comment 36 ABCC8 p.Arg74Trp We show that R74W reduces the stability of TMD0 protein and thus physical coupling between TMD0 and Kir6.2. Login to comment 37 ABCC8 p.Glu128Lys ABCC8 p.Glu128Trp In contrast, E128K disrupts functional coupling between TMD0 and Kir6.2 by abrogating the effects of SUR1 on channel response to PIP2. Moreover, the mutation E128W leads to spontaneous current inactivation that can be prevented or reversed by PIP2. Login to comment 70 ABCC8 p.Arg74* ABCC8 p.Glu128* Online supplemental material Fig. S1 shows the surface expression of R74X and E128X full-length KATP channels, as quantified by chemiluminescence after overnight pretreatment with 300 µM tolbutamide to rescue channel trafficking. Fig. S2 includes a protein sequence alignment performed by PRALINE (Simossis and Heringa, 2005) of TMD0 of SUR1 for human, hamster, dog, and zebrafish, and human SUR2. Login to comment 78 ABCC8 p.Arg74* ABCC8 p.Arg74Lys Surface expression for every R74X-mutant tested, except the charge-conserving R74K mutation, was significantly reduced to <40% of WT (Fig. 1 B). Login to comment 100 ABCC8 p.Arg74Trp ABCC8 p.Arg74Trp ABCC8 p.Arg74Asp ABCC8 p.Arg74Asp Because of significantly reduced surface expression in most mutants, cells were pretreated with 300 µM tolbutamide overnight, which partially corrects the trafficking defect caused by R74W, as reported previously (Yan et al., 2004, 2007; Pratt et al., 2009), as well as all other R74 mutations, with the exception of R74D (Fig. S1). Login to comment 101 ABCC8 p.Arg74Asp Because R74D had no detectable surface expression before or after tolbutamide exposure, it was not analyzed. Login to comment 107 ABCC8 p.Arg74Lys R74K, which had surface expression at 74 ± 4% of WT by chemiluminescence (Fig. 1 B), showed both upper and lower glycosylation bands. Login to comment 108 ABCC8 p.Arg74Ala ABCC8 p.Arg74Tyr In contrast, R74A and R74Y (2 ± 1 and 4 ± 2% surface expression by chemiluminescence, respectively) showed mostly core-glycosylated band, with very little complex-glycosylated SUR1 protein. Login to comment 109 ABCC8 p.Arg74* ABCC8 p.Arg74* ABCC8 p.Glu128* ABCC8 p.Glu128* Figure 1. Expression studies of fSUR1 R74X and E128X KATP channels. Login to comment 114 ABCC8 p.Arg74* ABCC8 p.Glu128* (C) Representative immunoblots using anti-SUR1 antibody to detect expression of fSUR1 protein in cells cotransfected with Kir6.2 and either fSUR1 R74X (top) or E128X (bottom) cDNAs. Login to comment 116 ABCC8 p.Arg74* ABCC8 p.Glu128* Note the blots shown for R74X and E128X are from two separate experiments; therefore, signal intensity should be compared within each blot only. Login to comment 117 ABCC8 p.Glu128* (D) Chemiluminescence assays performed to assess surface expression of E128X KATP channels. Login to comment 119 ABCC8 p.Arg74* R74 mutations in TMD0 constructs reduce steady-state protein levels and disrupt surface expression of mini-KATP channels Assessing the specific effects of R74X mutations on TMD0 is possible using a truncated SUR1 construct (amino acids 1-198). Login to comment 120 ABCC8 p.Arg74* Steady-state levels of total cellular fTMD0 harboring R74X were assessed via Western blot. Login to comment 121 ABCC8 p.Arg74Lys R74K had the highest level, with 70% of WT; all other R74 substitutions (alanine, phenylalanine, tyrosine, and tryptophan) resulted in significantly decreased TMD0 levels, with <25% of WT (Fig. 3, A and B), possibly a result of increased degradation. Login to comment 122 ABCC8 p.Arg74Trp ABCC8 p.Arg74Lys Next, we examined the surface expression of R74W and R74K fTMD0 when coexpressed with Kir6.2C36 using immunofluorescent obtain the IC50 for ATP inhibition (Fig. 2 B). Login to comment 123 ABCC8 p.Arg74Lys ABCC8 p.Arg74Lys ABCC8 p.Arg74Lys As with trafficking, the charge-conserving mutant R74K had minimal effect on ATP inhibition (IC50 = 18 ± 2 and 17 ± 1 µM for WT and R74K, respectively). Login to comment 126 ABCC8 p.Arg74* ABCC8 p.Arg74* ABCC8 p.Glu128* ABCC8 p.Glu128* Figure 2. Functional studies of fSUR1 R74X and E128X KATP channels. Login to comment 127 ABCC8 p.Arg74* ABCC8 p.Glu128* (A) Representative traces from inside-out voltage clamp experiments performed in COSm6 cells transfected with WT Kir6.2 and WT, R74X (top), or E128X (bottom) SUR1. Login to comment 129 ABCC8 p.Glu128Cys Bars: horizontal, 10 s; vertical, 1,000 pA for WT and E128C, and 200 pA for the rest. Login to comment 130 ABCC8 p.Arg74* ABCC8 p.Glu128* (B and C) ATP sensitivity expressed as half-maximal inhibitory concentration (IC50) for each R74X (B) and E128X (C) mutant. Login to comment 136 ABCC8 p.Arg74* ABCC8 p.Glu128* Broadly, E128X mutants showed greater surface expression than R74X mutants. Login to comment 137 ABCC8 p.Glu128* ABCC8 p.Glu128Arg Of the E128X channels tested, charge-reversal mutations (E128R and K) impeded surface expression most strongly (8 and 6% of WT, respectively). Login to comment 138 ABCC8 p.Glu128Asp Interestingly, the charge-conserving mutation E128D also showed relatively poor surface expression, only 38% that of WT channels. Login to comment 139 ABCC8 p.Glu128* Next, ATP-induced inhibition of E128X-mutant channels was examined. Login to comment 141 ABCC8 p.Arg74Lys No surface fTMD0 was detected above background signal using anti-FLAG antibody for either mutant construct, even though the R74K mutation exhibited the greatest total TMD0 level (70% of WT; Fig. 3 B); in contrast, surface staining of cells expressing WT mini-KATP channels was clearly visible (Fig. 3 E). Login to comment 142 ABCC8 p.Arg74Trp ABCC8 p.Arg74Lys Consistently, in single-channel recordings from cells coexpressing R74K or R74W fTMD0 with Kir6.2C36, no channel kinetics distinct from those characteristic of channels formed by Kir6.2C36 alone were observed (unpublished data). Login to comment 144 ABCC8 p.Arg74* ABCC8 p.Arg74* ABCC8 p.Glu128* ABCC8 p.Glu128* Figure 3. Biochemical and immunostaining studies of TMD0 harboring select R74X or E128X mutations. Login to comment 145 ABCC8 p.Arg74* ABCC8 p.Glu128* (A and C) Representative immunoblots of FLAG-tagged SUR1 TMD0 constructs with either R74X (A) or E128X (C) mutations expressed in COSm6 cells. Login to comment 147 ABCC8 p.Arg74* ABCC8 p.Glu128* (B and D) Densitometry analysis was performed on the blots in A and C to quantify R74X (B) or E128X (D) fTMD0 protein levels relative to WT fTMD0. Login to comment 149 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Lys (E) Cells cotransfected with WT, R74W, R74K, or E128K fTMD0 and Kir6.2C36 were probed with -FLAG antibody 48 h after transfection to detect surface expression of mini-KATP channels. Login to comment 150 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp ABCC8 p.Arg74Lys A rim of surface staining was detected in WTand E128K-transfected cells (green, inset images), but not in either R74W- or R74K-transfected cells. Login to comment 154 ABCC8 p.Glu128* To further investigate how E128 contributes to KATP channel physiology, we analyzed E128X TMD0 proteins and the resulting mini-KATP channels when coexpressed with Kir6.2C36. Login to comment 156 ABCC8 p.Glu128Lys ABCC8 p.Arg74* Also in contrast to R74X, E128K fTMD0 was detected at the cell surface of COSm6 cells when cotransfected with Kir6.2C36 (Fig. 3 E), indicating that the formation of mini-KATP channels is suitable for electrophysiological analysis. Login to comment 157 ABCC8 p.Glu128Lys Previously, we reported that full-length KATP channels with the E128K mutation in SUR1 have decreased intrinsic Po (Po = 0.18 vs. 0.63 for WT; Pratt et al., 2009). Login to comment 158 ABCC8 p.Glu128Lys ABCC8 p.Glu128* We tested whether the same would be true for E128K mini-KATP channels, which would be a clear indication of each E128X mutant (Fig. S1), followed by a 2-h washout before recording. Login to comment 161 ABCC8 p.Arg74* ABCC8 p.Glu128* E128 mutations disrupt functional coupling between TMD0 and Kir6.2 in mini-KATP channels E128X substitutions affect KATP channel surface expression and ATP sensitivity in a pattern distinct from R74X substitutions. Login to comment 162 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys This suggests different roles of the two Figure 4. E128K mini-KATP channels have decreased intrinsic Po. Login to comment 163 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu (A) Representative traces from inside-out voltage clamp recordings made from COSm6 cells transfected with Kir6.2C36 (denoted as Kir6.2C) alone or with WT, E128K, or F132L fTMD0 to form mini-KATP channels. Login to comment 168 ABCC8 p.Glu128Lys The average Po of E128K mini-KATP channels is significantly different from WT mini-KATP channels and Kir6.2C36 channels (*, P < 0.05; Student`s t test). Login to comment 172 ABCC8 p.Phe132Leu Again, the F132L mini-channels were included as a control because they have increased Po and decreased ATP sensitivity compared with WT mini-KATP channels (Proks et al., 2007). Login to comment 173 ABCC8 p.Glu128Lys ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu As predicted, the E128K mini-channels were inhibited to a similar extent as Kir6.2C36 channels (85%), whereas the WT and F132L mini-channels were inhibited by only 52 and 15%, respectively (compare Fig. 4, B and C). Login to comment 174 ABCC8 p.Glu128Lys This result provides further evidence that E128K directly uncouples functional interactions between TMD0 and Kir6.2, thus preventing the influence TMD0 has on the Po and ATP sensitivity of Kir6.2C36 channels. Login to comment 175 ABCC8 p.Glu128Lys E128K attenuates the response of full-length channels to PIP2 The intrinsic Po of Kir channels is thought to be determined by channel interactions with membrane phosphoinositides, in particular, PIP2: the stronger the interactions, the higher the Po (Logothetis et al., 2007; Xie et al., 2007). Login to comment 178 ABCC8 p.Glu128Lys In inside-out patches, the application of PIP2 to the bath solution rapidly increases WT channel that E128K disrupts functional coupling between TMD0 and Kir6.2. Login to comment 179 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys Single-channel recordings made from COSm6 cells cotransfected with E128K fTMD0 and Kir6.2C36 showed that the average intrinsic Po of E128K mini-KATP channels was, indeed, significantly lower than WT mini-channels (Po = 0.17 ± 0.04 for E128K vs. 0.60 ± 0.06 for WT; P < 0.05) (Fig. 4, A and B). Login to comment 180 ABCC8 p.Glu128Lys Further, the E128K mini-channels exhibited short bursts of activity separated by relatively long closures, distinguishing them from WT mini-channels and channels composed of Kir6.2C36 alone. Login to comment 181 ABCC8 p.Phe132Leu ABCC8 p.Phe132Leu Mini-KATP channels with the F132L mutation were used as a positive control; F132L was identified in patients with severe neonatal diabetes and has been shown to increase intrinsic Po in full-length and mini-KATP channels (Proks et al., 2006, 2007). Login to comment 182 ABCC8 p.Glu128Lys These results demonstrate that E128K in both full-length SUR1 and TMD0 alone reduces functional coupling to Kir6.2 with regard to intrinsic Po and single-channel kinetics. Login to comment 185 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys Because E128K abrogated the effect of TMD0 on the Po of Kir6.2, we predicted that mini-KATP channels with the E128K mutation will have an ATP sensitivity similar to Kir6.2C36 channels. Login to comment 186 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Phe132Leu WT, E128K, or F132L Figure 5. E128K full-length KATP channels have decreased PIP2 response. Login to comment 187 ABCC8 p.Glu128Lys (A) Traces of inside-out voltage clamp recordings from COSm6 cells transfected with Kir6.2 and WT (top) or E128K (bottom) SUR1. Login to comment 189 ABCC8 p.Glu128Lys Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 100 pA. Zero current is indicated by the dotted lines. Login to comment 191 ABCC8 p.Glu128Lys Error bars represent SEM; n = 8 (WT) and 12 (E128K) patches (*, P < 0.05; Student`s t test). Login to comment 192 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys current increase upon PIP2 exposure in E128K is striking when one considers that the starting Po of E128K is so much lower than WT, such that WT Po has the potential to increase from 0.6 to 1.0, whereas E128K Po has the potential to increase from 0.2 to 1.0. Login to comment 193 ABCC8 p.Glu128Lys To further illustrate the differential response in WT and E128K channels with respect to PIP2-induced current amplitude increase, we excised membrane patches into Kint plus 1 mM Mg2+ to induce rundown, such that WT channel Po before PIP2 exposure was substantially reduced (Fig. 6). Login to comment 195 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys In contrast, successive exposures of E128K channels to PIP2 after rundown also led to recovery of channel activity, but the current amplitude never reached that seen before rundown, such that the fold change in current amplitude relative to the initial current after successive 30-s PIP2 exposures was only 0.5 for E128K (0.4 ± 0.1, 0.5 ± 0.1, and 0.5 ± 0.1 after one, two, and three PIP2 exposures, respectively; Fig. 6 B). Login to comment 196 ABCC8 p.Glu128Lys Interestingly, we noted that the E128K channels were activity to maximal Po and decreases channel sensitivity to ATP inhibition. Login to comment 198 ABCC8 p.Glu128Lys We hypothesize that SUR1 modulates the Po of Kir6.2 via TMD0 by enhancing channel response to PIP2. Moreover, E128 is essential for this modulation, as shown by the diminished effect E128K TMD0 had on the Po of Kir6.2 (Fig. 4). Login to comment 199 ABCC8 p.Glu128Lys Accordingly, we predicted that the E128K channel will have reduced sensitivity to PIP2. Login to comment 200 ABCC8 p.Glu128Lys Indeed, we found that KATP channels harboring the E128K mutation were much less responsive to PIP2, both in terms of increase in current amplitude (i.e., Po) and decrease in ATP inhibition compared with WT. Login to comment 201 ABCC8 p.Glu128Lys The effect of PIP2 on the current amplitude of WT and E128K channels was first examined using our standard recording protocol in which membrane patches were excised into Kint bath solution containing 1 mM EDTA, which prevents channel rundown (Lin et al., 2003). Login to comment 202 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys After 30 s of exposure to 5 µM PIP2, WT current increased 1.53 ± 0.12-fold relative to current before PIP2 exposure, whereas E128K channel current increased only 1.07 ± 0.10-fold (Fig. 5, A and B). Login to comment 204 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys The lack of Figure 6. Rundown induced by 1 mM Mg2+ enhances the difference between WT and E128K response to PIP2 stimulation. Login to comment 205 ABCC8 p.Glu128Lys (A) Representative inside-out patch voltage clamp recordings from COSm6 cells transfected with WT (top) or E128K (bottom) KATP channels. Login to comment 206 ABCC8 p.Glu128Lys Patches were pulled into Kint plus 1 mM MgCl2 for 1 min to induce rundown to accentuate the differential response between WT and E128K channels; EDTA was not included in any of the solutions. Login to comment 208 ABCC8 p.Glu128Lys Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 100 pA. Zero current is indicated by the dotted lines. Login to comment 212 ABCC8 p.Glu128Lys Error bars represent SEM. n = 5-8 for each data point (*, P < 0.05; Student`s t test comparing WT and E128K for each point analyzed). Login to comment 215 ABCC8 p.Glu128Trp Inactivation of E128W channels could be recovered by exposure to high concentrations of ATP (1, 3, or 5 mM) in a time-dependent manner, such that lengthening the time of ATP ex-posure resulted in more current when the patch was reexposed to nucleotide-free solution (Fig. 8, A and B). Login to comment 216 ABCC8 p.Glu128Trp Further, exposure of the E128W patches to PIP2 slowed or reversed the inactivation as well as potentiated the ATP-induced "resetting" of channel activity (Fig. 8 C). Login to comment 217 ABCC8 p.Glu128Trp These results indicate that E128W causes KATP channel inactivation by destabilizing PIP2-KATP channel interactions, and that ATP exposure re- est-ablishes the interactions as seen in the transient channel activity when the inhibitory effect of ATP is removed. Login to comment 220 ABCC8 p.Glu128Lys This effect was also abrogated in the E128K mutant. Login to comment 221 ABCC8 p.Glu128Lys Patches containing WT or E128K channels were excised into Kint/EDTA solution and then exposed to differing concentrations of ATP (Fig. 7 A). Login to comment 223 ABCC8 p.Glu128Lys However, in E128K channels, exposure to PIP2 had little effect on ATP inhibition (Fig. 7, A and C). Login to comment 224 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys Collectively, the reduced response to PIP2 in the E128K channels resembles that seen in Kir6.2C36 channels, providing evidence that E128K disrupts the ability of SUR1 to enhance channel response to PIP2. Login to comment 225 ABCC8 p.Glu128Trp ABCC8 p.Glu128Trp The E128W mutation in SUR1 causes inactivation in full-length channels that can be recovered by PIP2 and by exposure and subsequent removal of ATP Additional evidence supporting the involvement of E128 in mediating channel response to PIP2 came from the intriguing phenotype observed with the E128W mutation. Login to comment 226 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys As mentioned above, the mutation of E128 to a trypto- Figure 7. Decrease of ATP sensitivity in response to PIP2 is abrogated by the E128K mutation. Login to comment 227 ABCC8 p.Glu128Lys (A) Representative inside-out patch voltage clamp recordings from COSm6 cells transfected with WT (top) or E128K (bottom) KATP channels. Login to comment 228 ABCC8 p.Glu128Lys Bars: WT, 1 min and 1,000 pA; E128K, 1 min and 500 pA. Zero current is indicated by the dotted lines. Login to comment 229 ABCC8 p.Glu128Lys ABCC8 p.Glu128Lys (B and C) Current inhibition by ATP was assessed using several ATP concentrations (0.01, 0.1, and 1 mM) before PIP2 exposure (filled black symbols) and after three 30-s exposures to 5 µM ATP (open gray symbols) for WT (B) and E128K (C) channels. Login to comment 238 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Mutations in TMD0 such as R74W and E128K offer potential for probing the molecular basis of TMD0-Kir6.2 signaling, as they cause dramatic impediments to channel trafficking and gating. Login to comment 239 ABCC8 p.Glu128Lys ABCC8 p.Arg74Trp Here, we studied full-length and mini-KATP channels to learn more about the functional roles of R74 and E128. We show that R74W and E128K disrupt channel function by different mechanisms. Login to comment 243 ABCC8 p.Glu128Trp ABCC8 p.Glu128Trp Although the exact location of R74 with respect to the membrane has not Figure 8. E128W causes KATP channel inactivation that can be recovered by ATP exposure and subsequent removal of ATP and can be reversed by PIP2. Login to comment 249 ABCC8 p.Glu128Trp (B) The ability of high concentrations (1-5 mM) of ATP to reset the E128W KATP channel is illustrated by this scatter plot. Login to comment 254 ABCC8 p.Glu128Trp (C) Representative trace showing that exposure of the E128W KATP channels to 5 µM PIP2 (striped bars) decreased and reversed inactivation as well as increased the efficacy of ATP (black lines, 5 mM) to reset the channel. Login to comment 257 ABCC8 p.Arg74* nately, however, R74X TMD0 mutants failed to express at the cell surface when coexpressed with Kir6.2C36, making it difficult to address the question of whether the gating defects in full-length channels are caused by changes in the functional coupling between TMD0 and Kir6.2 or are a consequence of altered influences by SUR1 structures outside TMD0. Login to comment 258 ABCC8 p.Arg74Lys ABCC8 p.Arg74Lys It may seem surprising that although total R74K TMD0 protein remains at near WT levels (Fig. 3, A and B), no R74K mini-channels were observed at the cell surface either by immunostaining or single-channel recording. Login to comment 259 ABCC8 p.Arg74Lys That the R74K mutation has a more pronounced effect on the surface expression of mini-KATP versus full-length channels (i.e., no detectable surface expression of mini-channels [Fig. 3 C] vs. 80% of WT for full-length channels [Fig. 1, B and C]) suggests a role for extra-TMD0 regions of SUR1 in channel protein folding and assembly. Login to comment 261 ABCC8 p.Arg74* Thus, the here comes into play when hypothesizing the structural changes R74X substitutions have on TMD0 conformation. Login to comment 263 ABCC8 p.Arg74* ABCC8 p.Arg74Asp ABCC8 p.Arg74Glu Accordingly, with the exception of lysine, all other amino acids used in the R74X screen (A, H, C, W, F, Y, L, D, or E) are predicted to shift the boundary of TM1 and TM2 (as well as TM3 for R74D and R74E) by TOPCONS (Fig. S2). Login to comment 264 ABCC8 p.Arg74* The decrease in full-length KATP channel surface expression of R74X mutants (Fig. 1) and steady-state protein levels of TMD0 (Fig. 3) is consistent with these substitutions having an effect on protein structure and stability. Login to comment 265 ABCC8 p.Arg74* ABCC8 p.Arg74Asp Sulfonylureas acting as chemical chaperones partially overcome the trafficking defect in R74X full-length KATP channels, with the exception of R74D (Fig. S1). Login to comment 269 ABCC8 p.Glu128Lys (A) Cartoon illustrating proposed physical relationships between SUR1, Kir6.2, and PIP2 in the WT, E128K mutant, and the Kir6.2C36 channels. Login to comment 270 ABCC8 p.Glu128Trp (B) The E128W mutation destabilizes the channel in the SUR1-Kir6.2-coupled, PIP2-bound open state, leading to channel inactivation. Login to comment 275 ABCC8 p.Glu128Lys Mini-KATP channels with the E128K mutation also follow this relationship; i.e., they show reduced Po and increased ATP inhibition relative to WT mini-channels (similar to that seen for Kir6.2C36). Login to comment 276 ABCC8 p.Glu128Lys Although the inverse relationship between Po and ATP sensitivity might predict that in full-length channels the E128K mutant should show increased ATP sensitivity relative to WT, this is not the case. Login to comment 277 ABCC8 p.Glu128Lys Rather, E128K full-length channels actually have reduced ATP inhibition. Login to comment 278 ABCC8 p.Glu128Lys Thus, by abolishing the ability of TMD0 to enhance Kir6.2 response to PIP2, the E128K mutation abrogates the ability of structures downstream of TMD0 to confer increased ATP sensitivity to Kir6.2. Login to comment 279 ABCC8 p.Glu128Lys Future identification of SUR1 residues responsible for increasing ATP sensitivity of the channel will be critical for understanding precisely how E128K uncouples SUR1 from Kir6.2 with respect to regulation by both PIP2 and ATP. Login to comment 280 ABCC8 p.Glu128Trp ABCC8 p.Glu128Trp A novel KATP channel inactivation mechanism revealed by the E128W mutation and structural implications The importance of E128 in the structural and functional integrity of KATP channels is further accentuated by the inactivation phenotype of the E128W mutant. Login to comment 282 ABCC8 p.Glu128Trp First, current inactivation induced by E128W is distinct from rundown. Login to comment 283 ABCC8 p.Glu128Trp Second, inactivation caused by E128W is prevented or reversed by PIP2. Login to comment 287 ABCC8 p.Glu128Trp That inactivation induced by E128W is overcome by PIP2 lends strong support to our proposal that E128 plays a critical role in the stabilization of Kir6.2-PIP2 interactions by SUR1. Login to comment 288 ABCC8 p.Glu128Trp ABCC8 p.Arg74Lys In addition, the ATP-dependent recovery from inactivation in the E128W mutant suggests that conformational changes in Kir6.2 tertiary structure of R74K TMD0 and its ability to assemble with Kir6.2 to form channels may be contingent upon the SUR1 structures downstream of TMD0. Login to comment 289 ABCC8 p.Arg74* ABCC8 p.Glu128* Role of E128 Full-length channels bearing E128X mutations are in general expressed at a higher level at the cell surface than R74X channels (Fig. 1). Login to comment 290 ABCC8 p.Glu128Lys Moreover, mutations at residue 128 have less effect on TMD0 protein levels, and E128K TMD0 can form mini-channels with Kir6.2C36 that traffic to the cell surface (Fig. 3). Login to comment 293 ABCC8 p.Glu128Lys However, the E128K mutation in both full-length and mini-KATP channels shifts the Po and ATP sensitivity toward those seen in Kir6.2C36 channels, indicating that E128 contributes to functional coupling with Kir6.2. Login to comment 294 ABCC8 p.Glu128Lys Strikingly, E128K renders full-length channels much less sensitive to PIP2 stimulation, resembling that seen in Kir6.2C36 channels. Login to comment 306 ABCC8 p.Glu128Lys The detrimental effect of E128K on channel interaction with PIP2 therefore offers a clear explanation for how the mutation reduces Po in full-length and mini-KATP channels. Login to comment 307 ABCC8 p.Glu128Lys However, the explanation for the effects of E128K on ATP sensitivity in full-length and mini-KATP channels is more complex. Login to comment 310 ABCC8 p.Glu128Trp Finally, our discovery of the E128W inactivation mutation provides novel insight into the structural relationship between channel subunits as a function of ATP and PIP2. Login to comment 347 ABCC8 p.Glu128Trp The similar gating properties seen in E128W and Kir6.2 inactivation mutations predicted to alter inter-Kir6.2 subunit interfaces imply that these mutations disrupt normal channel gating via a converging mechanism that is sensitive to both PIP2 and ATP. Login to comment 352 ABCC8 p.Glu128Trp The finding is especially provocative when considered in the context of the SUR1-Kir6.2 KATP channel complex because it opens up the possibility that SUR1 can modulate Kir6.2 conduction primarily through cytoplasmic domain interfaces, such as that mediated by E128. We propose that in KATP channels, SUR1 stabilizes a Kir6.2 structure in the PIP2-bound open state via cytoplasmic interactions, and that the inactivation phenotype of E128W represents transition from a SUR1-coupled conformation to Kir6.2 structures lacking the stabilizing effect of SUR1, as seen in Kir6.2C36. Login to comment 353 ABCC8 p.Glu128Trp ATP binding is envisioned to cause a conformational switch in Kir6.2, such that channels can enter a PIP2-bound open state once ATP is removed.Thus,inthecaseofKir6.2inactivationmutations, ATP reactivates the channel by reestablishing Kir6.2 subunit interface (Lin et al., 2003), and in the case of E128W, by reestablishing SUR1-Kir6.2 interactions necessary for stabilizing channel opening, albeit only briefly (see Fig. 9 B). Login to comment 354 ABCC8 p.Glu128Trp Increasing the concentration of PIP2 in the membrane is expected to shift the equilibrium toward the PIP2-bound open state by mass action, thereby slowing and reversing inactivation as well as boosting the ATP-induced resetting of the channel in both Kir6.2 and E128W-SUR1 inactivation mutants (Figs. Login to comment 391 ABCC8 p.Phe132Leu Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome. Login to comment