ABCMdb

PMID: 17673962

Zhou JJ, Fatehi M, Linsdell P
Direct and indirect effects of mutations at the outer mouth of the cystic fibrosis transmembrane conductance regulator chloride channel pore.
J Membr Biol. 2007 Apr;216(2-3):129-42. Epub 2007 Aug 3., [PubMed]

Sentences

No. Mutations Sentence Comment

23 ABCC7 p.Arg334Lys However, all mutations studied, including the charge-conservative R334K, lead to significant weakening of Au(CN)2 À binding (Gong & Linsdell, 2003a), which is inconsistent with an electrostatic interaction. Login to comment 28 ABCC7 p.Arg334Lys However, single-channel recording showed that all mutations at this site, including the charge-conservative R334K, lead to a dramatic decrease in the amplitude of unitary currents carried by ClÀ efflux through the pore (Gong & Linsdell, 2004). Login to comment 68 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Whether studied under conditions of low (Fig. 1a) or high (Fig. 1b) extracellular ClÀ concentration, Pt(NO2)4 2À block is significantly weakened in both K95Q and R303Q (Figs. 1c, 2). Login to comment 70 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Furthermore, a double mutant in which both of these positive charges are neutralized (K95Q/R303Q) generated currents that were almost completely insensitive to the blocking effects of Pt(NO2)4 2À (Fig. 1), consistent with the overall channel-blocking effects of Pt(NO2)4 2À ions being the result of interactions with both of these purportedly independent sites (St. Aubin et al., 2007). Login to comment 71 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln In spite of the weakened blocking effects of intracellular Pt(NO2)4 2À seen in both K95Q and R303Q, block of each of these two mutants was still significantly weakened by extracellular ClÀ ions (Figs. 1c, 2). Login to comment 72 ABCC7 p.Arg334Gln Neutralization of a third pore-forming positive charge, in the R334Q mutant, was also associated with significant weakening of the blocking effects of internal Pt(NO2)4 2À (Figs. 1, 2). Login to comment 73 ABCC7 p.Arg334Cys This is consistent with previously reported weakening of intracellular Pt(NO2)4 2À block in R334C (Gong & Linsdell, 2003b). Login to comment 74 ABCC7 p.Arg334Gln ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln However, unlike K95Q and R303Q, the R334Q mutant weakened Pt(NO2)4 2À block at low, but not high, extracellular ClÀ concentration (Fig. 2). Login to comment 75 ABCC7 p.Arg334Gln This difference likely results from the fact that the R334Q mutation also apparently removes the sensitivity of block to extracellular ClÀ concentration (Figs. 1c, 2). Login to comment 76 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln As described above, weakened block by intracellular Pt(NO2)4 2À ions in K95Q and R303Q is consistent with Fig. 1 Removal of positive charges in the pore weakens block by intracellular Pt(NO2)4 2À ions. Login to comment 81 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln ABCC7 p.Lys95Gln ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln ABCC7 p.Arg303Gln Mean of data from three to eight patches. Fitted lines are to equation 1, with the following parameters: wild type 4 mM external ClÀ , Kd(0) = 85.8 lM, zd = À0.201; wild type 154 mM external ClÀ , Kd(0) = 387 lM, zd = À0.344; K95Q 4 mM external ClÀ , Kd(0) = 403 lM, zd = À0.130; K95Q 154 mM external ClÀ , Kd(0) = 978 lM, zd = À0.227; R303Q 4 mM external ClÀ , Kd(0) = 300 lM, zd = À0.096; R303Q 154 mM external ClÀ , Kd(0) = 904 lM, zd = À0.197; R334Q 4 mM external ClÀ , Kd(0) = 286 lM, zd = À0.330; R334Q 154 mM external ClÀ , Kd(0) = 256 lM, zd = À0.307. Login to comment 82 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Because block was so weak at this concentration, data for the K95Q/R303Q double mutant were not fitted current models of the pore that place these two positively charged amino acid residues within the pore inner vestibule. Login to comment 85 ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys Figure 3 shows the blocking effects of internally applied Pt(NO2)4 2À in six different channel mutants (R334C, R334E, R334H, R334K, R334L, R334Q) under conditions of both low (Fig. 3a) and high (Fig. 3b) extracellular ClÀ concentration. Login to comment 90 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu With low extracellular ClÀ concentrations, the Kd for Pt(NO2)4 2À block (at 0 mV) was significantly increased in all six R334 mutants studied (Fig. 5a), although it is clear that R334C and R334E had far greater effects on Kd compared to other amino acid substitutions. Login to comment 91 ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys With elevated extracellular ClÀ , the Kd(0) was significantly increased only in R334C and R334E; not significantly altered in R334K, R334L and R334Q; and significantly decreased in R334H (Fig. 5b). Login to comment 93 ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334His These R334 mutations also exhibited a weakened sensitivity of blocker voltage dependence (quantified as -zd, Fig. 5) to external ClÀ concentration (Fig. 5c), although because of the small magnitude of this effect only R334C, R334H and R334Q reached a level of statistical significance (Fig. 5c). Login to comment 106 ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys Comparison of the mean Kd estimated for suramin (at 0 mV) shows that R334C, R334E, R334K, R334L and R334Q were all associated with weakened suramin block, with only R334H failing to significantly affect suramin block (Fig. 7). Login to comment 107 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu Suramin block was particularly weakened in R334C and R334E (Figs. 6, 7) such that the profiles of mutation effects on block by internal Pt(NO2)4 2À and suramin are very similar. Login to comment 110 ABCC7 p.Arg334Gln As shown in Figure 8, although block by internal Pt(NO2)4 2À is significantly weakened in R334Q, this blocking effect is still sensitive to the presence of Pt(NO2)4 2À ions in the extracellular solution. Login to comment 111 ABCC7 p.Arg334Gln In wild type, addition of 8 mM Pt(NO2)4 2À to the extracellular solution increased mean Kd(0) for intracellular Pt(NO2)4 2À approximately 2.7-fold without significantly altering -zd, while in R334Q this concentration of extracellular Pt(NO2)4 2À increased mean Kd(0) 2.5-fold, again with no significant change in -zd (Fig. 8c). Login to comment 112 ABCC7 p.Arg334Gln Thus, the R334Q mutation practically abolishes the dependence of intracellular Pt(NO2)4 2À block on external ClÀ (Figs. 1c, 2, 5c) without significantly altering its dependence on external Pt(NO2)4 2À (Fig. 8). Login to comment 129 ABCC7 p.Arg334Leu ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys ABCC7 p.Arg334Lys Mean of data from three to eight patches. Fitted lines are to equation 1 as described in Figure 1 for wild type and R334Q and with the following parameters for other channel variants: R334C 4 mM external ClÀ , Kd(0) = 1362 lM, zd = À0.295; R334C 154 mM external ClÀ , Kd(0) = 836 lM, zd = À0.219; R334E 4 mM external ClÀ , Kd(0) = 759 lM, zd = À0.376; R334E 154 mM external ClÀ , Kd(0) = 564 lM, zd = À0.173; R334H 4 mM external ClÀ , Kd(0) = 140 lM, zd = À0.166; R334H 154 mM external ClÀ , Kd(0) = 119 lM, zd = À0.149; R334K 4 mM external ClÀ , Kd(0) = 143 lM, zd = À0.314; R334K 154 mM external ClÀ , Kd(0) = 317 lM, zd = À0.374; R334L 4 mM external ClÀ , Kd(0) = 176 lM, zd = À0.258; R334L 154 mM external ClÀ , Kd(0) = 284 lM, zd = À0.366 extracellular Pt(NO2)4 2À by normalizing current amplitude at the hyperpolarized extreme of the voltage range studied, -80 mV (Fig. 10b). Login to comment 136 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Example macroscopic current-voltage relationships for wild type and each R334 mutant studied, as well as mutations that neutralize positive charges involved in binding of intracellular Pt(NO2)4 2À (K95Q, R303Q), are compared in Figure 11a. Login to comment 139 ABCC7 p.Arg334Lys However, it can be seen that for all R334 mutations except the charge-conservative R334K, current-voltage relationship shape was not strongly altered by the presence of Pt(NO2)4 2À in the extracellular solution. Login to comment 140 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Both K95Q and R303Q are associated with outward rectification under control conditions; this rectification was apparently weakened in the presence of extracellular Pt(NO2)4 2À , most likely due to voltage-dependent current inhibition, which was most prominent at depolarized voltages. Login to comment 141 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln The mean effects of 10 mM Pt(NO2)4 2À in each channel variant, analyzed as described above for wild type (Fig. 10c), are illustrated in Figure 11b (for R334 mutants) and 11c (for K95Q and R303Q). Login to comment 142 ABCC7 p.Arg334Lys ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Considering only the data at +80 mV (Fig. 11d), where block of wild-type CFTR is strongest (Figs. 9, 10), the blocking effects of Pt(NO2)4 2À are slightly (but significantly) weakened in K95Q, R303Q and R334K (p < 0.05) but practically abolished in all other R334 mutants (p < 0.0005). Login to comment 143 ABCC7 p.Arg334Lys ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln In fact, only wild type, R334K, K95Q and R303Q - those mutants that retain a positive charge at position 334 - were significantly affected by 10 mM Pt(NO2)4 2À according to this analysis (as illustrated by the daggers in Fig. 11d, p < 0.001), whereas all mutants associated with removal of the positive charge at R334 showed no significant differences in the absence or presence of external Pt(NO2)4 2À (p > 0.15). Login to comment 149 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Our present results suggest that block by intracellular Pt(NO2)4 2À involves interactions with positively charged amino acid side chains in the wide inner vestibule of the pore since mutations that remove these positive charges (K95Q, R303Q) lead to significant weakening of Pt(NO2)4 2À block (Figs. 1, 2). Login to comment 150 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Furthermore, removal of both positive charges (in the K95Q/R303Q double mutant) gave rise to a channel that was almost completely resistant to the blocking effects of internal Pt(NO2)4 2À (Fig. 1), suggesting that both positive charges contribute to Pt(NO2)4 2À binding in the pore inner vestibule. Login to comment 159 ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334Cys ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys These plots represent mean data from four to seven patches. Fitted lines are to equation 1 with the following parameters: wild type, Kd(0) = 2.51 lM, zd = À0.042; R334C, Kd(0) = 18.5 lM, zd = À0.056; R334E, Kd(0) = 25.0 lM, zd = À0.107; R334H, Kd(0) = 3.10 lM, zd = À0.085; R334K, Kd(0) = 6.31 lM, zd = À0.232; R334L, Kd(0) = 4.08 lM, zd = À0.061; R334Q, Kd(0) = 6.64 lM, zd = À0.239 with our previous suggestion that intracellular Au(CN)2 À blocks the channel by interacting directly with R334, several reasons prompt us to suggest that Pt(NO2)4 2À does not interact directly with the arginine side chain at this position. Login to comment 161 ABCC7 p.Arg334Lys Second, Pt(NO2)4 2À block is significantly weakened in all of the six R334-mutated variants studied, including the charge-conservative R334K (Fig. 5a). Login to comment 162 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu Thus, while Pt(NO2)4 2À block is particularly weak in R334C and R334E (Figs. 3-5), there is no strong correlation between the apparent affinity of Pt(NO2)4 2À block and the nature of the side chain present at position 334. Login to comment 166 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu The similar effects of mutations on block by intracellular suramin and intracellular Pt(NO2)4 2À - in particular, the strong effects of R334C and R334E on the inhibitory effects of both blockers - suggest that these mutations affect suramin block and Pt(NO2)4 2À block by a common mechanism. Login to comment 171 ABCC7 p.Arg334Gln a Example leak-subtracted macroscopic current-voltage relationships recorded with 8 mM K2Pt(NO2)4 2À present in the extracellular solution for wild type (left) and R334Q (right), recorded before (control) and after (+ Pt[NO2]4) addition of 300 lM K2Pt(NO2)4 to the intracellular solution. Login to comment 173 ABCC7 p.Arg334Gln ABCC7 p.Arg334Gln Mean of data from three to eight patches. Fitted lines are to equation 1 with the following parameters: wild type (), Kd(0) = 85.8 lM, -zd = 0.201; wild type (d), Kd(0) = 205 lM, - zd = 0.248; R334Q (), Kd(0) = 286 lM, -zd = 0.330; R334Q (d), Kd(0) = 704 lM, -zd = 0.401. c Mean Kd(0) and -zd calculated under these conditions, without extracellular Pt(NO2)4 2À (white bars) and with 8 mM Pt(NO2)4 2À (black bars). Login to comment 178 ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Thus, all R334 mutants studied disrupted the dependence of intracellular Pt(NO2)4 2À block on extracellular ClÀ ions (Fig. 5), whereas Pt(NO2)4 2À block of both K95Q and R303Q remained Cl-dependent (Figs. 1, 2). Login to comment 185 ABCC7 p.Arg334Lys In contrast, the charge-conservative R334K mutant showed only very slightly weakened block by external Pt(NO2)4 2À (Fig. 11d). Login to comment 200 ABCC7 p.Arg334Lys Our results are also entirely consistent with extracellular Pt(NO2)4 2À ions screening the surface charge contributed by R334 (or by the substituted lysine in R334K), resulting in loss of electrostatic attractive forces on extracellular ClÀ ions and reduced ClÀ entry into the pore at depolarized voltages. Login to comment 214 ABCC7 p.Arg334Gln Furthermore, we suggest that external Pt(NO2)4 2À and external ClÀ destabilize the binding of internal blockers by different mechanisms since the effects of ClÀ , but not Pt(NO2)4 2À , are lost in R334Q. Login to comment 218 ABCC7 p.Arg334Lys As a result, these direct effects show a strong dependence on side chain charge, being strongly disrupted in all mutants except R334K. Login to comment 221 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu However, disruption of the effects of intracellular blockers is particularly pronounced in R334C and R334E (Figs. 5, 7). Login to comment 222 ABCC7 p.Arg334Cys ABCC7 p.Arg334Glu Currently, we have no explanation as to why R334C and R334E have so much more dramatic effects on block by intracellular Pt(NO2)4 2À and suramin than other R334 mutations. Login to comment 226 ABCC7 p.Arg334Lys ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln Note the change in current rectification induced by extracellular Pt(NO2)4 2À in wild type, R334K, K95Q and R303Q but not other R334 mutants. Login to comment 227 ABCC7 p.Arg334Cys b,c Mean fraction of control normalized current remaining in the presence of 10 mM extracellular Pt(NO2)4 2À at different membrane potentials for different channel variants: b wild type (d), R334C (. Login to comment 228 ABCC7 p.Arg334Leu ABCC7 p.Arg334Gln ABCC7 p.Arg334His ABCC7 p.Arg334Glu ABCC7 p.Arg334Lys ABCC7 p.Lys95Gln ABCC7 p.Arg303Gln ), R334E (5), R334H (j), R334K (), R334L (h), R334Q (u); c wild type (d), K95Q (m), R303Q (Å). Login to comment