ABCMdb

PMID: 15361410

Liu X, Zhang ZR, Fuller MD, Billingsley J, McCarty NA, Dawson DC
CFTR: a cysteine at position 338 in TM6 senses a positive electrostatic potential in the pore.
Biophys J. 2004 Dec;87(6):3826-41. Epub 2004 Sep 10., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Thr338Cys Xenopus oocytes expressing T338C CFTR exhibited pH-dependent changes in gCl and I-V shape that were specific to the substituted cysteine. Login to comment 2 ABCC7 p.Thr338Cys The apparent pKa of T338C CFTR was more acidic than that expected for a cysteine or similar simple thiols in aqueous solution. Login to comment 4 ABCC7 p.Thr338Cys The relative rates of chemical modification of T338C CFTR by MTSET1 and MTSESÿ were also altered by the charge at 334. Login to comment 53 ABCC7 p.Thr338Cys In studies of T338C CFTR, the pipette solution contained 196 mM NMDG-Cl, 2 mM MgCl2, and 5 mM HEPES HemiNa, pH 7.4; 5 mM MES was used in place of HEPES when a pH of 6 was desired for the pipette solution. Login to comment 54 ABCC7 p.Thr338Cys In studies of T338C CFTR, the pipette solution contained 196 mM NMDG-Cl, 2 mM MgCl2, and 5 mM HEPES HemiNa, pH 7.4; 5 mM MES was used in place of HEPES when a pH of 6 was desired for the pipette solution. Login to comment 64 ABCC7 p.Thr338Ala The single channel current for T338A CFTR was measured using 150 mM symmetrical [Clÿ ]. Login to comment 69 ABCC7 p.Thr338Cys Recordings of single, T338C channels in experiments involving covalent modification posed additional challenges for two reasons. Login to comment 72 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys Finally, preliminary experiments indicated that, in contrast to results obtained previously with R334C CFTR, reaction of T338C channels with MTSET1 actually reduced single-channel conductance, rendering the resulting single-channel currents more difficult to discriminate from the small (0.1-0.3 pA) background Clÿ channels sometimes seen in patches from oocytes. Login to comment 79 ABCC7 p.Thr338Cys Recording conditions were designed to maximize the likelihood of recording T338C single channels in each of three states: unmodified; MTSET1 modified; and MTSET1 modified, but subsequently exposed to 2-ME to reverse the modification (see text). Login to comment 92 ABCC7 p.Thr338Cys We interpreted the titration behavior of the conductance due to T338C CFTR as reflecting the pH-dependent change in the time-averaged, partial negative charge on any single thiolate anion. Login to comment 94 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338His ABCC7 p.Thr338His ABCC7 p.Arg334* For ease of comparison, in T338C/R334X (X ¼ A or E) CFTRs and T338H/R334C CFTRs in which the cysteine was always blocked by reaction with MTS reagents or NEM, the titration curves were expressed in a normalized form. Login to comment 103 ABCC7 p.Thr338Cys RESULTS The conductance of oocytes expressing T338C CFTR is highly sensitive to changes in bath pH Testing the hypothesis that a cysteine at position 338 resides within the conduction pathway required that we alter the charge at this locus. Login to comment 104 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys In previous experiments using R334C CFTR we found that charge change could be effected by means of covalent modification with thiol-directed reagents like MTSET1 or MTSESÿ , or by using changes in bath pH to alter the partial negative charge on the thiolate anion (Smith et al., 2001). Login to comment 105 ABCC7 p.Arg334Cys In previous experiments using R334C CFTR we found that charge change could be effected by means of covalent modification with thiol-directed reagents like MTSET1 or MTSES , or by using changes in bath pH to alter the partial negative charge on the thiolate anion (Smith et al., 2001). Login to comment 106 ABCC7 p.Thr338Cys Fig. 2 A contains a plot of gCl at Vm ¼ Erev versus time from a representative experiment (n ¼ 5) designed to test the pH-sensitivity of T338C CFTR. Login to comment 107 ABCC7 p.Thr338Cys Fig. 2 A contains a plot of gCl at Vm &#bc; Erev versus time from a representative experiment (n &#bc; 5) designed to test the pH-sensitivity of T338C CFTR. Login to comment 113 ABCC7 p.Thr338Cys It can be seen that as the bath pH became more acidic the I-V plot changed in two ways: gCl at Vm ¼ Erev FIGURE 2 The pH-induced change in conductance of oocytes expressing T338C CFTR. Login to comment 114 ABCC7 p.Thr338Cys It can be seen that as the bath pH became more acidic the I-V plot changed in two ways: gCl at Vm &#bc; Erev FIGURE 2 The pH-induced change in conductance of oocytes expressing T338C CFTR. Login to comment 120 ABCC7 p.Thr338Cys (B) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338C CFTR. Login to comment 121 ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys (C) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338A CFTR. Login to comment 122 ABCC7 p.Thr338Ala (C) The I-V plots obtained at pH 9 (dashed line), 7.4 (dotted line), and 6 (solid line) from an oocyte expressing T338A CFTR. Login to comment 124 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys Changing the bath pH had essentially no effect on the conductances of oocytes expressing T338A CFTR (Fig. 2 C, n ¼ 3), nor did the same maneuver alter the conductances of oocytes expressing T338S (Fig. 3) or wt CFTR (Smith et al., 2001), consistent with the idea that the pH-dependent change in conductance of T338C CFTR was due to the titration of the cysteine substituted at 338. Login to comment 125 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys As an additional test of the hypothesis that the pH-induced response seen in T338C CFTR was due to the titration of the engineered cysteine, we exposed oocytes expressing T338C CFTR to NEM, a reagent that forms a thioether bond with the cysteine, and thereby blocks titration of the thiol group. Login to comment 126 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys As summarized in Fig. 3, acidifying the bathing solution (pH 7.4-6) induced an ;68% (612) increase in conductance in oocytes expressing T338C CFTR whereas alkalinizing the bath (pH 7.4-9) decreased the conductance by ;80% (64). Login to comment 127 ABCC7 p.Thr338Cys As summarized in Fig. 3, acidifying the bathing solution (pH 7.4-6) induced an ;68% (612) increase in conductance in oocytes expressing T338C CFTR whereas alkalinizing the bath (pH 7.4-9) decreased the conductance by ;80% (64). Login to comment 128 ABCC7 p.Thr338Cys Pre-exposure to 1 mM MTSET1 or 1 mM MTSESÿ also prevented the response of T338C CFTR conductance to a change in bath pH (X. Liu and D. C. Dawson, unpublished observation). Login to comment 129 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Changes in anion conduction brought about by titration of T338C CFTR are consistent with the predictions of a simplified charged-vestibule model We compared the effect of charge changes at 338 to the predictions of simplified charged-vestibule models used previously to analyze the influence of charge at position 334 (Smith et al., 2001). Login to comment 130 ABCC7 p.Thr338Cys Changes in anion conduction brought about by titration of T338C CFTR are consistent with the predictions of a simplified charged-vestibule model We compared the effect of charge changes at 338 to the predictions of simplified charged-vestibule models used previously to analyze the influence of charge at position 334 (Smith et al., 2001). Login to comment 136 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Single-channel conductance of T338C CFTR is pH-sensitive Fig. 5 A contains examples of T338C CFTR single-channel currents recorded from detached inside-out patches with the pH of the pipette solution (containing 1 mM 2-ME) buffered to either pH 6 or pH 7.4. Login to comment 137 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Single-channel conductance of T338C CFTR is pH-sensitive Fig. 5 A contains examples of T338C CFTR single-channel currents recorded from detached inside-out patches with the pH of the pipette solution (containing 1 mM 2-ME) buffered to either pH 6 or pH 7.4. Login to comment 143 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys We speculate that these events might be attributed to the opening of some endogenous channels or alternatively, to T338C CFTR channels that were in oxidation FIGURE 3 The pH-sensitivity of T338C CFTR conductance was due to the titration of the cysteine thiol. Login to comment 144 ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Oocytes expressing T338C or T338S CFTR were first exposed to a stimulatory cocktail containing 10 mM Isop and 1 mM IBMX at pH 7.4. Login to comment 145 ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys Oocytes expressing T338C or T338S CFTR were first exposed to a stimulatory cocktail containing 10 mM Isop and 1 mM IBMX at pH 7.4. Login to comment 148 ABCC7 p.Thr338Cys Premodification by NEM prevented the large pH-induced response seen in T338C CFTR conductance. Login to comment 149 ABCC7 p.Thr338Ser ABCC7 p.Thr338Cys T338S CFTR exhibited no pH-sensitivity. Login to comment 150 ABCC7 p.Thr338Ser T338S CFTR exhibited no pH-sensitivity. Login to comment 155 ABCC7 p.Thr338Ala Single-channel recording also indicated that the conductance of T338A CFTR channels was not sensitive to changes in bath pH (Fig. 5 C, inset). Login to comment 156 ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys It is also of interest to note that the ratio of the single channel conductance at pH 6 and pH 7.4 for T338C CFTR was ;1.8, a value comparable to the ratio observed for the macroscopic conductances (1.7). Login to comment 157 ABCC7 p.Thr338Cys It is also of interest to note that the ratio of the single channel conductance at pH 6 and pH 7.4 for T338C CFTR was ;1.8, a value comparable to the ratio observed for the macroscopic conductances (1.7). Login to comment 164 ABCC7 p.Thr338Ala No pH-induced change in Po* was observed in T338A CFTR, which averaged 0.85 6 1.2 at pH 6 and averaged 0.74 6 0.15 at pH 7.4 ([ATP] ¼ 1 mM). Login to comment 165 ABCC7 p.Thr338Ala No pH-induced change in Po* was observed in T338A CFTR, which averaged 0.85 6 1.2 at pH 6 and averaged 0.74 6 0.15 at pH 7.4 ([ATP] &#bc; 1 mM). Login to comment 166 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The macroscopic conductance due to T338C CFTR exhibits a pKa that is more acidic than expected for a simple thiol in free solution Shown in Fig. 7 is a representative titration curve obtained by measuring the conductance of an oocyte expressing T338C CFTR at different values of bath pH (n ¼ 5). Login to comment 167 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The macroscopic conductance due to T338C CFTR exhibits a pKa that is more acidic than expected for a simple thiol in free solution Shown in Fig. 7 is a representative titration curve obtained by measuring the conductance of an oocyte expressing T338C CFTR at different values of bath pH (n &#bc; 5). Login to comment 169 ABCC7 p.Thr338Cys (A) Using the continuum model, the I-V curve corresponding to pH 6 (open squares) obtained from an oocyte expressing T338C CFTR was first fitted by fixing Co at zero, resulting in a PCl of 3.51 3 10ÿ7 cm3 /s, a Ci of 42 mM, and a Ci of 20.9 mV. Login to comment 170 ABCC7 p.Thr338Cys (A) Using the continuum model, the I-V curve corresponding to pH 6 (open squares) obtained from an oocyte expressing T338C CFTR was first fitted by fixing Co at zero, resulting in a PCl of 3.51 3 107 cm3 /s, a Ci of 42 mM, and a Ci of 20.9 mV. Login to comment 177 ABCC7 p.Arg334Cys This procedure attributed the effects of MTSET1 and MTSESÿ on the conductance of R334C CFTR to a change in Co of 45 mV and ÿ30 mV, respectively, a prediction somewhat different from that reported by Smith et al., 2001, in which the effects of MTSET1 and MTSESÿ were attributed to a change in Co of 50 mV and ÿ10 mV, respectively.) Login to comment 178 ABCC7 p.Arg334Cys This procedure attributed the effects of MTSET1 and MTSES on the conductance of R334C CFTR to a change in Co of 45 mV and 30 mV, respectively, a prediction somewhat different from that reported by Smith et al., 2001, in which the effects of MTSET1 and MTSES were attributed to a change in Co of 50 mV and 10 mV, respectively.) Login to comment 184 ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp To investigate the effect of charge at position 334 on the titration behavior of T338C CFTR, we examined the conductance of oocytes expressing double mutants, T338C/R334A, T338C/R334E, and T338C/R334D CFTR. Login to comment 185 ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp Shown in Fig. 8 A are representative titration curves for the conductance for T338C CFTR and two of these double mutants (n ¼ 5 each). Login to comment 186 ABCC7 p.Arg334Ala ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Neutralizing the charge at 334 (R334A) resulted in a pKa that was more than one pH unit more basic (8.78 6 0.03, n ¼ 4) than that determined for T338C CFTR. Login to comment 187 ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n ¼ 4 and 8.96 6 0.08 for T338C/R334E CFTR, n ¼ 5). Login to comment 188 ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp The substitution of acidic residues, however, did not result in a large additional shift of the apparent pKa to more alkaline values (8.84 6 0.05 for T338C/R334D CFTR, n &#bc; 4 and 8.96 6 0.08 for T338C/R334E CFTR, n &#bc; 5). Login to comment 190 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338His ABCC7 p.Thr338His To test the generality of titration results obtained with T338C CFTR, we compared the titration behavior of T338H CFTR with that of a double mutant, T338H/R334C, in which it was possible to change the charge at position 334 by means of chemical modification. Login to comment 191 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338His ABCC7 p.Thr338His ABCC7 p.Thr338His Summarized in Fig. 8 B are the results obtained from oocytes expressing either T338H or chemically modified T338H/R334C CFTR (n ¼ 3-4 for each mutant). Login to comment 192 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His ABCC7 p.Thr338His ABCC7 p.Thr338His Shown are sample titration curves for T338H and T338H/R334C CFTRs in which the cysteine was modified by MTSET1 , MTSESÿ , and NEM (neutral). Login to comment 193 ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His Shown are sample titration curves for T338H and T338H/R334C CFTRs in which the cysteine was modified by MTSET1 , MTSES , and NEM (neutral). Login to comment 194 ABCC7 p.Thr338His The conductance of oocytes expressing T338H CFTR was essentially not titratable, as if the pKa of the histidine was more acidic than 4.0. Login to comment 195 ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His Similarly, the conductance due to MTSET1 -modified T338H/R334C CFTR was not very sensitive to pH titration. Login to comment 196 ABCC7 p.Arg334Cys ABCC7 p.Thr338His Similarly, the conductance due to MTSET1 -modified T338H/R334C CFTR was not very sensitive to pH titration. Login to comment 197 ABCC7 p.Thr338Cys (A) Examples of single-channel current records obtained from inside-out patches detached from oocytes expressing T338C CFTR. Login to comment 198 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys (B) The i-V plot for T338C CFTR. Login to comment 199 ABCC7 p.Thr338Cys (B) The i-V plot for T338C CFTR. Login to comment 205 ABCC7 p.Thr338Ala (Inset) T338A CFTR conductance was insensitive to changes in bath pH. Login to comment 206 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala Shown are examples of single-channel i-V plots obtained from inside-out patches detached from oocytes expressing T338A CFTR at pH 6 (solid circles) and pH 7.4 (shaded triangles). Login to comment 207 ABCC7 p.Thr338Ala ABCC7 p.Thr338Ala All the recordings for T338A CFTR were done in the presence of 150 mM symmetrical [Clÿ ]. Login to comment 208 ABCC7 p.Thr338Ala ABCC7 p.Arg334Cys ABCC7 p.Thr338His NEM- or MTSESÿ -modified T338H/R334C CFTR were more titratable and the apparent pKa values were shifted toward more basic values. Login to comment 209 ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His ABCC7 p.Thr338His Because the range of pH values tolerated by oocytes was limited, the apparent pKa values could not be estimated accurately for T338H and T338H/ R334C variants. Login to comment 210 ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His Because the range of pH values tolerated by oocytes was limited, the apparent pKa values could not be estimated accurately for T338H and T338H/ R334C variants. Login to comment 211 ABCC7 p.Thr338Cys Rates of covalent modification of T338C CFTR by MTSET1 and MTSES2 are consistent with a model featuring a positive vestibule potential If, as suggested by the shift in the apparent pKa of a cysteine or a histidine at 338, this portion of the CFTR pore is characterized by a positive electrostatic potential, the potential should also affect the relative rates of covalent modification by MTSET1 and MTSESÿ (Stauffer and Karlin, 1994; Pascual and Karlin, 1998; Karlin and Akabas, 1998). Login to comment 212 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Fig. 9 contains the results of experiments in which the time course of modification was measured at pH 7.4 for oocytes expressing either T338C CFTR or a double mutant FIGURE 6 Alteration of charge at the engineered cysteine at position 338 did not affect channel gating. Login to comment 213 ABCC7 p.Thr338Cys Fig. 9 contains the results of experiments in which the time course of modification was measured at pH 7.4 for oocytes expressing either T338C CFTR or a double mutant FIGURE 6 Alteration of charge at the engineered cysteine at position 338 did not affect channel gating. Login to comment 216 ABCC7 p.Thr338Cys FIGURE 7 Titration of conductance due to T338C CFTR. Login to comment 217 ABCC7 p.Thr338Cys FIGURE 7 Titration of conductance due to T338C CFTR. Login to comment 221 ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338His ABCC7 p.Thr338His FIGURE 8 The pH-induced changes in the conductances of oocytes expressing T338C/R334A, T338C/R334E, T338H, or T338H/R334C CFTR. Login to comment 222 ABCC7 p.Arg334Cys ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338His ABCC7 p.Thr338His (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment 223 ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys (A) Sample titration curves of conductances of oocytes expressing T338C CFTR (solid circles), T338C/R334A (open squares), or T338C/ R334E CFTRs (shaded triangles). Login to comment 225 ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His (B) Sample titration curves for T338H (open triangle) and T338H/R334C CFTR modified by MTSET1 (solid circles), MTSESÿ (open circles), and NEM (shaded triangles). Login to comment 226 ABCC7 p.Arg334Cys ABCC7 p.Thr338His ABCC7 p.Thr338His (B) Sample titration curves for T338H (open triangle) and T338H/R334C CFTR modified by MTSET1 (solid circles), MTSES (open circles), and NEM (shaded triangles). Login to comment 228 ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp in which the arginine at 334 was replaced by aspartic acid (T338C/R334D CFTR). Login to comment 229 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp Note that covalent modification of T338C CFTR with either reagent decreased the macroscopic conductance. Login to comment 230 ABCC7 p.Thr338Cys Note that covalent modification of T338C CFTR with either reagent decreased the macroscopic conductance. Login to comment 232 ABCC7 p.Thr338Cys Single-channel recordings (see below) showed that modification of T338C CFTR by MTSET1 reduced single-channel conductance, as expected if the ethyl(trimethylammonium) moiety partially obstructs anion flow through the pore. Login to comment 233 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The rate of modification of T338C CFTR by MTSESÿ exceeded that for MTSET1 , a result that is even more remarkable given the fact that the intrinsic rate of reaction of MTSESÿ with simple thiols is ;12-fold less than that for MTSET1 , due to the electrostatic interaction of the thiolate anion and the MTSESÿ during thiol-disulfide exchange (Karlin and Akabas, 1998). Login to comment 234 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp Introduction of negative charge at 334 (in T338C/R334D) reversed the relative reaction rates so that modification by MTSET1 was more rapid. Login to comment 235 ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp Introduction of negative charge at 334 (in T338C/R334D) reversed the relative reaction rates so that modification by MTSET1 was more rapid. Login to comment 236 ABCC7 p.Thr338Cys The time constants for MTSET1 and MTSESÿ modification of T338C CFTR ([MTS] ¼ 25 mM) averaged 64.5 6 2.2 s (n ¼ 3) and 11.3 6 1.9 s (n ¼ 3), respectively. Login to comment 237 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp The time constants for MTSET1 and MTSESÿ modification of T338C/R334D CFTR ([MTS] ¼ 25 mM) averaged 39.8 6 15.8 s (n ¼ 3) and 641 6 27.7 s (n ¼ 3), respectively. Login to comment 238 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp Calculations based on a simple kinetic model for the ratio of the rates of reaction of MTSET1 and MTSESÿ , including a correction for the difference in the intrinsic rates of the MTS-thiolate reactions (see Discussion), suggested that charged reagents modifying T338C CFTR sensed an electrostatic potential that was ;54 mV positive with respect to the bath. Login to comment 239 ABCC7 p.Thr338Cys Calculations based on a simple kinetic model for the ratio of the rates of reaction of MTSET1 and MTSES , including a correction for the difference in the intrinsic rates of the MTS-thiolate reactions (see Discussion), suggested that charged reagents modifying T338C CFTR sensed an electrostatic potential that was ;54 mV positive with respect to the bath. Login to comment 240 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Covalent modification of T338C CFTR by MTSET1 adds a positive charge, but also partially obstructs the pore The reduction of T338C CFTR macroscopic conductance by MTSET1 suggested that at this locus, the deposition of the ethyl(trimethylammonium) moiety might partially block the pore. Login to comment 241 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Direct evidence for pore obstruction by MTSET1 was obtained by recording T338C CFTR single-channel currents before and after modification. Login to comment 242 ABCC7 p.Thr338Cys Direct evidence for pore obstruction by MTSET1 was obtained by recording T338C CFTR single-channel currents before and after modification. Login to comment 247 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp The effective concentration of MTSET1 in the pipette likely varied because the half-life of MTSET1 is only ;10 min at room temperature according to Stauffer FIGURE 9 Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET1 (n ¼ 3) and MTSESÿ (n ¼ 3). Login to comment 248 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp The effective concentration of MTSET1 in the pipette likely varied because the half-life of MTSET1 is only ;10 min at room temperature according to Stauffer FIGURE 9 Representative time course of modification of T338C CFTR and T338C/R334D CFTR conductance by MTSET1 (n &#bc; 3) and MTSES (n &#bc; 3). Login to comment 249 ABCC7 p.Thr338Cys (A) After activation, oocytes expressing T338C CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSESÿ (:). Login to comment 250 ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSESÿ (d). Login to comment 251 ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp (B) After activation, oocytes expressing T338C/R334D CFTR were first exposed to reducing agents (2-ME) followed by a wash and were then exposed to 25 mM MTSET1 (s) or MTSES (d). Login to comment 265 ABCC7 p.Thr338Cys This result is consistent with the notion that the 0.2-pA event represented MTSET1 -modified T338C CFTR channels. Login to comment 266 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Based on the single-channel amplitude observed under reducing conditions, it seems likely that the 0.2-pA events included a small contribution from a background channel or a terminal oxidation state of T338C CFTR; see Materials and Methods. Login to comment 267 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The fact that the modification of the T338C CFTR channel was not always complete may reflect the acidic pH as well as some degradation of the MTSET1 . Login to comment 268 ABCC7 p.Thr338Cys The fact that the modification of the T338C CFTR channel was not always complete may reflect the acidic pH as well as some degradation of the MTSET1 . Login to comment 271 ABCC7 p.Thr338Cys As an additional test of the effect of MTSET1 we examined changes in single-channel current amplitude in experiments in which the recording pipette was backfilled with MTSET1 to FIGURE 10 MTSET1 modification of T338C CFTR single-channel conductance. Login to comment 272 ABCC7 p.Thr338Cys As an additional test of the effect of MTSET1 we examined changes in single-channel current amplitude in experiments in which the recording pipette was backfilled with MTSET1 to FIGURE 10 MTSET1 modification of T338C CFTR single-channel conductance. Login to comment 278 ABCC7 p.Thr338Cys The results suggested a 75% decrease in single channel current at Vm ¼ ÿ100 mV at pH 6 due to MTSET1 -induced modification of T338C CFTR. Login to comment 279 ABCC7 p.Thr338Cys The results suggested a 75% decrease in single channel current at Vm &#bc; 100 mV at pH 6 due to MTSET1 -induced modification of T338C CFTR. Login to comment 289 ABCC7 p.Thr338Cys If, as suggested by single-channel recordings, MTSET1 partially obstructs the pore of T338C CFTR, we reasoned that the net effect of MTSET1 on macroscopic conductance might consist of two components: one due to obstruction and another due to the change in charge. Login to comment 290 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys In previous experiments using R334C CFTR (Smith et al., 2001) we showed that, in general, the total charge change due to covalent modification was the sum of the charge added by the deposited group and the pH-dependent charge on the thiolate that is neutralized in the formation of a mixed disulfide bond. Login to comment 291 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys The results shown in Fig. 12 compare the effectsof covalent modificationof T338C CFTR at pH 6 and pH 9, values chosen such that the partial charge on the cysteine thiolate would vary from near zero (pH 6) to near ÿ1 (pH 9). Login to comment 292 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys At pH 9, MTSET1 increased T338C CFTR conductance by 50-110% (84 6 15, n ¼ 3) whereas at pH 6, the same treatment reduced the conductance due to T338C CFTR by 20-80% (n ¼ 16). Login to comment 293 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys At pH 9, MTSET1 increased T338C CFTR conductance by 50-110% (84 6 15, n &#bc; 3) whereas at pH 6, the same treatment reduced the conductance due to T338C CFTR by 20-80% (n &#bc; 16). Login to comment 295 ABCC7 p.Thr338Cys This apparent twofold difference in efficacy is FIGURE 11 Real-time modification of T338C-CFTR channels activated by PKA and ATP in an excised, inside-out patch. Login to comment 296 ABCC7 p.Thr338Cys This apparent twofold difference in efficacy is FIGURE 11 Real-time modification of T338C-CFTR channels activated by PKA and ATP in an excised, inside-out patch. Login to comment 297 ABCC7 p.Thr338Cys During the course of the recording, MTSET1 diffused to the pipette tip, and modified the T338C-CFTR channels there. Login to comment 298 ABCC7 p.Thr338Cys During the course of the recording, MTSET1 diffused to the pipette tip, and modified the T338C-CFTR channels there. Login to comment 302 ABCC7 p.Arg334Cys The blocking effect of MTSET1 at position 338 contrasts to the effect of the same reagent at position 334, where MTSET1 increased the conductance of R334C CFTR by ;45% (616) at pH 6 and by ;92% (612) at pH 9 (Smith et al., 2001). Login to comment 304 ABCC7 p.Arg334Cys The blocking effect of MTSET1 at position 338 contrasts to the effect of the same reagent at position 334, where MTSET1 increased the conductance of R334C CFTR by ;45% (616) at pH 6 and by ;92% (612) at pH 9 (Smith et al., 2001). Login to comment 305 ABCC7 p.Thr338Cys Cheung and Akabas (1996) reported that T338C CFTR did not react with externally applied MTSET1 or MTSESÿ and concluded that the residue did not lie on the outward-facing, water-accessible surface of the protein. Login to comment 307 ABCC7 p.Thr338Cys Cheung and Akabas (1996) reported that T338C CFTR did not react with externally applied MTSET1 or MTSES and concluded that the residue did not lie on the outward-facing, water-accessible surface of the protein. Login to comment 313 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys The observation that modification of T338C CFTR by MTSET1 produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn. Login to comment 314 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The pKa of T338C CFTR is consistent with a model in which T338C resides in the pore where it senses a positive electrostatic potential The pH titration of the conductances of oocytes expressing T338C CFTR was consistent with the stabilizing effect of a nearby positive charge on the thiolate anion, and the behavior of double mutants suggested that one source of this charge could be R334. Login to comment 315 ABCC7 p.Arg334Cys ABCC7 p.Thr338Cys The observation that modification of T338C CFTR by MTSET1 produced a net reduction in single-channel conductance, whereas similar modification of R334C CFTR increased single-channel conductance, is compatible with the notion that the pore narrows over the length of the helical turn. Login to comment 316 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The pKa of T338C CFTR is consistent with a model in which T338C resides in the pore where it senses a positive electrostatic potential The pH titration of the conductances of oocytes expressing T338C CFTR was consistent with the stabilizing effect of a nearby positive charge on the thiolate anion, and the behavior of double mutants suggested that one source of this charge could be R334. Login to comment 318 ABCC7 p.Thr338Cys FIGURE 12 The pH-dependent effect of MTSET1 on whole-cell T338C CFTR conductance. Login to comment 320 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys (A) At pH 6, MTSET1 reduced the conductance due to T338C CFTR by variable amount (20-80%). Login to comment 322 ABCC7 p.Thr338Cys (A) At pH 6, MTSET1 reduced the conductance due to T338C CFTR by variable amount (20-80%). Login to comment 325 ABCC7 p.Thr338Cys In the case of T338C CFTR, although we lack the information as to a three-dimensional structure, we can nevertheless make use of some of the general principles that have emerged from the analysis of other proteins. Login to comment 327 ABCC7 p.Thr338Cys In the case of T338C CFTR, although we lack the information as to a three-dimensional structure, we can nevertheless make use of some of the general principles that have emerged from the analysis of other proteins. Login to comment 329 ABCC7 p.Thr338Cys The total potential, CT o ; for T338C CFTR, would also contain a pH-dependent component due to the cysteine thiolate at 338. Login to comment 330 ABCC7 p.Thr338Cys As indicated above, at alkaline pH, the charge at 338 would be likely to make a large contribution to CT o in T338C CFTR. Login to comment 331 ABCC7 p.Thr338Cys The total potential, CT o ; for T338C CFTR, would also contain a pH-dependent component due to the cysteine thiolate at 338. Login to comment 332 ABCC7 p.Thr338Cys As indicated above, at alkaline pH, the charge at 338 would be likely to make a large contribution to CT o in T338C CFTR. Login to comment 338 ABCC7 p.Thr338Cys The pKa values range from 8.3 for free cysteine to ;10.3 for methylthiol or ethylthiol, and would translate into values for Cq o ranging from 53 mV to 171 mV positive with respect to the bath using a pKa of 7.4 for T338C CFTR. Login to comment 340 ABCC7 p.Arg334Ala ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys A comparison of the apparent pKa of T338C CFTR with that of the double mutant, T338C/R334A, suggests that the amino acid substitution at position 334 shifted the pKa from ;7.4 to 8.8 or ;1.4 units. Login to comment 342 ABCC7 p.Arg334Ala ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys A comparison of T338C/R334A (pKa ¼ 8.8) with T338C/R334E (pKa ¼ 8.9), would suggest a further change in Cq o of ;ÿ6 mV. Login to comment 343 ABCC7 p.Thr338Cys The relative rates of modification of T338C CFTR by MTSET1 and MTSESÿ provide another estimate of Cq o (Stauffer and Karlin, 1994; Pascual and Karlin, 1998; Karlin and Akabas, 1998; Wilson et al., 2000; Elinder et al., 2001). Login to comment 344 ABCC7 p.Arg334Ala ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys A comparison of T338C/R334A (pKa &#bc; 8.8) with T338C/R334E (pKa &#bc; 8.9), would suggest a further change in Cq o of ;6 mV. Login to comment 345 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp The ratio of the rates of modification is given by Eq. 7 (see Supplementary Material), k MTSES k MTSET ¼ k MTSES i k MTSET i exp 2 F RT Cq o : (7) Using the ratio for kMTSES i =kMTSET i of 1/12 measured for 2-ME (Karlin and Akabas, 1998), the relative rates of modification for these two compounds yields a value for Cq o of 54 mV for T338C CFTR and ÿ4 mV for T338C/R334D CFTR, comparable to the values estimated from the shift in cysteine pKa resulting from the replacement of arginine by alanine or aspartic acid. Login to comment 347 ABCC7 p.Arg334Ala ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys ABCC7 p.Arg334Asp Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles. Login to comment 348 ABCC7 p.Arg334Ala ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The absolute values of Cq o must be interpreted with caution since we do not know to what extent structural differences between these two mutants might have resulted from the amino acid substitution (see below), but both of the measurements used to estimate the electrostatic potential of the vestibule indicated an asymmetry between the impact of basic and acidic residues at 334. Prediction of the electrostatic effects of R334 If we ignore the possible effects of structural changes in the CFTR protein produced by amino acid substitution, then the change in Cq o calculated from the difference in the pKa of T338C/R334 and T338C/R334A can be taken to be a crude measure of CR334 o ; the component of Cq o due to the native arginine, and we can compare the value derived experimentally with that predicted on the basis of first principles. Login to comment 356 ABCC7 p.Thr338Cys The results obtained using two rather different approaches are both consistent with the hypothesis that the observed shift in the apparent pKa of T338C CFTR could be the result of the electrostatic effect of R334. Login to comment 357 ABCC7 p.Thr338Cys The results obtained using two rather different approaches are both consistent with the hypothesis that the observed shift in the apparent pKa of T338C CFTR could be the result of the electrostatic effect of R334. Login to comment 360 ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys It would not be surprising to find that the thiolate- arginine ion pair of T338C/R334 was closer together than the thiolate-glutamic acid pair of T338C/R334E, even if there were no major change in structure that accompanied the mutations. Login to comment 361 ABCC7 p.Arg334Glu ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys It would not be surprising to find that the thiolate- arginine ion pair of T338C/R334 was closer together than the thiolate-glutamic acid pair of T338C/R334E, even if there were no major change in structure that accompanied the mutations. Login to comment