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Figure 1. Chemical structures for the thiol-specific reagents used in this study.
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Figure 2. MTS reagents did not alter the function of wt CFTR expressed in oocytes. Conductance (@Erev) is plotted versus time. After CFTR was activated by the application of 1 mM IBMX and 10 μM forskolin (hatched bar; see materials and methods), oocytes were exposed sequentially to 1 mM 2-ME (white bars and circles), 1 mM MTSET, MTSEA, and MTSES (black bars and circles).
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Figure 3. Comparison of the effects of MTSES, MTSET, and MTSEA on the conductance of oocytes expressing R334C, K335C, R347C, or R352C CFTR. Plotted are values for the percent change in oocyte conductance (Δg/ginitial × 100%) induced by each reagent (±SEM). Asterisk indicates significant difference from unmodified conductance, P < 0.05.
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Figure 4. MTS reagents did not discernibly alter the function of R347C CFTR expressed in oocytes. Protocol as in Fig. 2.
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Figure 5. MTSET and MTSES, polar thiol reagents, did not produce a discernible alteration in conductance of oocytes expressing R352C CFTR. Protocol as in Fig. 2. (A) Application of 1 mM 2-ME, 1 mM MTSET, or 1 mM MTSES produced no discernible change in conductance. 1 mM MTSEA produced a nearly fivefold increase in conductance, which spontaneously reversed when MTSEA was removed from the perfusate. (B) Addition of 1 mM MTSEA produced a sixfold increase in conductance of an oocyte expressing R352C CFTR not previously exposed to MTSET. The effect spontaneously reversed when MTSEA was removed from the perfusate. (C) MTSEA produced a twofold increase in conductance in an oocyte expressing R352Q CFTR, which spontaneously reversed when MTSEA was removed from the perfusate.
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Figure 6. Covalent modification of R334C CFTR was stable, reproducible, and varied with the electrostatic nature of the moiety covalently attached. Protocol as in Fig. 2. (A) Exposure to 1 mM MTSET resulted in doubling of the conductance, which remained elevated until the oocyte was exposed to 2-ME. 1 mM MTSEA resulted in an increase in conductance which was ∼70% of the increase elicited by MTSET, and was stable until the application of 2-ME. MTSES reduced the conductance to ∼30% of the unmodified level. Modification was stable and not readily reversed by the application of 1 mM 2-ME at pH 7.4. (B) Reapplication of MTSET reproduced the effects seen with the first application.
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Figure 7. R334C CFTR did not exhibit time-dependent current relaxations. (A) Currents evoked by stepping the potential from −100 to +60 mV for 234 ms from an oocyte expressing R334C CFTR after achieving steady-state activation with stimulatory cocktail and after a brief (3 min) exposure to 1 mM 2-ME. (B) Currents from the same oocyte after modification with 100 μM MTSET. (C) Currents from the same oocyte after a 12-min exposure to 1 mM 2-ME to reverse the MTSET modification followed by exposure to 100 μM MTSES. (D) I-V plots derived from the current traces shown in A–C: (closed circles) unmodified R334C CFTR; (open circles) MTSET modified R334C CFTR; and (closed triangles) MTSES modified R334C. There were readily detectable changes in the conductance and the shape of the I-V relation, but no shift in the reversal potential.
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Figure 8. Covalent modification of K335C CFTR altered both conductance and shape of the I-V relation. Protocol as in Fig. 2. (A) The oocyte was exposed to 2-ME (white bars) and MTS reagents (black bars). Positively charged reagents (MTSET and MTSEA) increased conductance and were readily reversed by 2-ME. The negatively charged reagent (MTSES) resulted in a ∼30% decrease in the conductance and was poorly reversible with 1 mM 2-ME at pH 7.4. (B) The rectification ratio (RR)—quantified as the slope conductance measured at +25 mV with respect to Erev divided by the slope conductance measured at Erev – 25 mV—plotted versus time for the same oocyte. RR of unity reflected a linear I-V relation and is noted with a dashed line. RR values greater or less than unity indicate outward and inward rectification, respectively. (C) I-V plots for K335C CFTR before and after application of MTS reagents. (solid line) Before modification; (dotted line) after treatment with MTSET; (dashed line) after treatment with MTSES. There were changes in the conductance and the shape of the current-voltage relation, but no shift in reversal potential.
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Figure 9. Changes in conductance and I-V shape induced by MTSET were not dependent on oocyte conductance or the activation state of CFTR. (A) Conductance of oocytes expressing R334C CFTR determined just after modification by MTSET plotted versus premodification conductance. Dashed line is least-squares fit to open circles, representing oocytes activated using 1 mM IBMX and 10 μM isoproterenol (slope equals 2.05, r = 0.987), dashed lines (95% confidence limits). (dark diamonds) Oocytes activated using 200 μM IBMX; (gray diamonds) oocytes after reversing MTSET modification, activation by 1 mM IBMX, and reexposure to MTSET. (B) Values of RR plotted versus corresponding oocyte conductance. (open and closed triangles) Oocytes activated using 1 mM IBMX, 10 μM isoproterenol. (closed and open diamonds) Oocytes exposed sequentially to 200 μM and 1 mM IBMX, in the absence of MTSET. (closed and open squares) Oocytes activated by 200 μM IBMX, 10 μM isoproterenol, exposed to MTSET and, after reversing MTSET (2-ME), activated again using 1 mM IBMX and reexposed to MTSET. Dashed lines are mean values of RR for modified and unmodified R334C CFTR.
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Figure 10. Covalent modification of R334C increased single-channel conductance. (A) Representative traces from an oocyte expressing R334C CFTR in the detached, inside-out patch configuration, Vm = −80 mV. The top trace was recorded before treatment with MTSET. The bottom trace was recorded from a separate patch on the same oocyte, after a 6-min exposure to 100 μM MTSET in the bath before establishing the seal. Channels were activated by the addition of 10 μM isoproterenol to the bath, before excising into the intracellular solution (see materials and methods) containing 1 mM MgATP and 50 U/ml PKA catalytic subunit. Bath and pipette solutions contained ∼210 mM Cl. The patch represented by the top trace contained only one active channel, whereas the lower patch contained two channels. Closed current level is indicated. (B) I-V plot for pooled single channel records (n = 4, pretreatment and n = 3, post-treatment). Standard errors are smaller than the symbols used. The solid lines are the best fit to the data by linear regression and correspond to a single-channel conductance of 1.2 pS for unmodified R334C CFTR (closed triangles) channels and 3.7 pS after MTSET modification (open triangles). MTSET did not alter single-channel conductance of wt CFTR (8.6 pS, open and closed circles). Inset shows open probability for R334C CFTR channels at Vm = −100 mV before and after treatment with MTSET (see materials and methods). There was no significant change in Po after treatment (P = 0.361, t test).
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Figure 11. Covalent modification of a single R334C CFTR channel in real time by including MTSET in the patch pipette. (A) Openings in a detached patch before and after modification which occurred at some time during the 75-s separating the last low amplitude opening and the first higher amplitude opening. Vm= −100 mV, [Cl]cyto = 300 mM and [Cl]pipette = 30 mM. (B) Openings from the same record with scale expanded. (C) Po determined before and after covalent modification for four, single R334C CFTR channels. Modification was assumed to have occurred at the midpoint between the last unmodified and first modified openings. Each value represents 3–5 min before and after modification.
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Figure 12. pH titration of oocytes expressing R334C CFTR altered both the conductance and the shape of the I-V relation. (A) I-V plot from an oocyte expressing R334C CFTR. After achieving steady-state activation in stimulatory cocktail at pH 7.4, the bath was acidified to pH 5.6 (solid line). Alkalinization of the bath to pH 8.0 (dotted line) or pH 9.7 (dashed line) decreased conductance and enhanced inward rectification without altering the reversal potential. (B) Percent increase in conductance with respect to the lowest conductance observed (most alkaline pH) plotted versus the bath pH for three different oocytes. was fitted to points from 5.6 to 10.0, and resulted in a mean pKa of 8.17 ± 0.10 (SEM) and a mean %gmax of 170 ± 6.9 (SEM). The solid line represents the Henderson-Hasselbach equation for a pKa of 8.17.
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Figure 13. Functional effect of covalent modification of R334C CFTR was dependent on the bath pH. Shown is the percent change in oocyte conductance due to covalent modification at either pH 9.0 or pH 6, as described in results. Also shown is a comparison of the measured pH-dependent difference in the effects of MTSET and MTSES compared with that predicted on the basis of a pKa for the cysteine of 8.17 and the assumption that conductance was a linear function of the charge at this locus (see Fig. 18 B).
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Figure 14. Modification of R334C CFTR with neutral reagents caused pH-dependent changes in conductance. (A) Change in conductance of oocytes expressing R334C CFTR when modified with either NEM or MTSHE at either pH 9.0 or pH 6. NEM behaved as expected for a neutral reagent whereas MTSHE, which is expected to be polar but uncharged, did not. (B) The results of similar experiments conducted using constructs in which the arginine at position 334 was replaced by a nonreactive amino acid (glutamine).
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Figure 15. pH titration of oocytes expressing R334H CFTR altered both the conductance and the shape of the I-V relation. (A) I-V plots from an oocyte expressing R334H CFTR. After achieving steady-state activation in stimulatory cocktail at pH 7.4 (solid line), acidification of the bath to pH 6.0 (dotted line) or pH 4.8 (dashed line) increased conductance and deprecated inward rectification without altering the reversal potential. (B) Percent increase in conductance, normalized to the lowest conductance observed (most alkaline pH), plotted versus the bath pH for four different oocytes. was fitted and resulted in a mean pKa of 5.68 ± 0.08 and a mean %gmax of 129 ± 15.5. The solid line represents the Henderson-Hasselbach equation for a pKa of 5.68.
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Figure 16. The shape of the I-V relation was correlated with the charge status of the amino acid substituted at positions 334 and 335. (A) RR, determined as described in materials and methods, is plotted as a function of the net charge of the amino acid at position 334 at pH 7.4 where glutamic acid (E) is assigned a value of −1, cysteine (C) is assigned a value of −0.12 based on the apparent pKa of 8.17 for R334C CFTR, histidine (H), alanine (A), and glutamine (Q) are neutral, and lysine (K) and arginine (R) are assigned a value of +1. (B) RR is plotted as a function of the net charge of the amino acid at position 335 where glutamic acid (E) and aspartic acid (D) were assigned a value of −1, alanine (A) was treated as neutral, cysteine (C) was also treated as neutral because K335C CFTR exhibited a smaller pH responses and its apparent pKa was not determined, and lysine (K) and arginine (R) were assigned a value of +1. The charge status of H and C is dependent on the bath pH so the assignment of a neutral value in a bath pH of 7.4 introduces a slight error. Solid lines are least-squares fits and dashed lines are 95% confidence limits.
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Figure 17. The effects of covalent modification of R334C CFTR could be simulated using charged vestibule models (see ). The data points shown are the same as in Fig. 7 D containing unmodified R334C CFTR (closed circles), MTSET-modified R334C (open circles), and MTSES-modified R334C (closed triangles). (A) Continuum model with PCl and Ψi fixed, allowing Ψo to vary (r2 = 0.993 for MTSET and 0.958 for MTSES). (B) Continuum model with PCl and Ψo fixed, allowing Ψi to vary (r2 = 0.879 for MTSET and 0.826 for MTSES). (C) 4-barrier, 3-well rate-theory model simulating a high affinity channel, apparent K1/2 = 38 mM. Barriers (RT) = 3.65, 5.7, 6, and 7.1; wells (RT) = −0.7, −2.8, and −2. (closed circles) Unmodified R334C CFTR, Ψo = 0. (open circles) MTSET-modified R334C CFTR, Ψo = + 50 mV. (closed triangles) MTSES-modified R334C CFTR, Ψo = −10 mV. (D) 4-barrier, 3-well rate-theory model simulating a low affinity channel, apparent K1/2 = 115 mM. Barriers (RT) = 3.8, 6, 7.5, and 7; wells (RT) = −0.1, −1.85, and −0.5. Symbols as in C for unmodified R334C CFTR (Ψo = 0), MTSET-modified R334C CFTR (Ψo = +50 mV), and MTSES-modified R334C CFTR (Ψo = −10 mV). (inset) Plots of percent increase (Δg/ginitial × 100%) in macroscopic CFTR conductance as function of bath Cl concentration for wt CFTR (open triangles) and unmodified R334C CFTR (closed circles).
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Figure 18. (A) The fractional changes in conductance and rectification ratio were correlated. Data points include charge changes brought about by thiol modification of R334C CFTR with positively charged reagents (open triangles), and negatively charged reagents (closed triangles), pH titration of R334C CFTR (closed circles), and pH titration of R334H CFTR (open circles). The solid line is the least-squares fit to the data. (B) Fractional change in conductance plotted versus the fractional change in elementary charge at position 334. Thiol modification of R334C CFTR with positively charged reagents (open triangles) was treated as adding a single positive charge; thiol modification of R334C with negatively charged reagents (closed triangles) was treated as adding a single negative charge; pH titration of R334H CFTR (open circles) was treated as adding a time-average positive charge determined by the bath pH, assuming a pKa of 5.68; and pH titration of R334C CFTR (closed circles) was treated as adding a time-average negative charge determined by the bath pH assuming a pKa of 8.17. Solid line is least-squares fit to the data, dashed lines are 95% confidence limits.
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Figure 19. Cartoon illustrating the features of the charged vestibule model.
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