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CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.

Smith SS, Liu X, Zhang ZR, Sun F, Kriewall TE, McCarty NA, Dawson DC - J. Gen. Physiol. (2001)

Bottom Line: Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot.Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability.The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, OR 97201, USA.

ABSTRACT
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

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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 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.

Mentions: It was of interest to compare the response to changes in bath pH of R334C CFTR with that of R334H CFTR. The side chain of histidine is expected to bear a time-average, partial positive charge, the magnitude of which will depend on the local pH and the pKa of the imidazole group in situ. An I-V plot from a representative experiment in which the pH of the solution bathing an oocyte expressing R334H CFTR was acidified from 7.4 to 6.02 and then 4.80 is shown in Fig. 15 A. Acidification of the bath from pH 7.4 to 6.02 led to an immediate increase in conductance and slight reduction in the inward rectification. Further acidification to pH 4.80 led to an additional increase in conductance and a nearly linear current-voltage relation without shifting the reversal potential. The apparent pKa for the conductance change was 5.68 ± 0.08 (n = 4; Fig. 15 B).


CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.

Smith SS, Liu X, Zhang ZR, Sun F, Kriewall TE, McCarty NA, Dawson DC - J. Gen. Physiol. (2001)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233702&req=5

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.
Mentions: It was of interest to compare the response to changes in bath pH of R334C CFTR with that of R334H CFTR. The side chain of histidine is expected to bear a time-average, partial positive charge, the magnitude of which will depend on the local pH and the pKa of the imidazole group in situ. An I-V plot from a representative experiment in which the pH of the solution bathing an oocyte expressing R334H CFTR was acidified from 7.4 to 6.02 and then 4.80 is shown in Fig. 15 A. Acidification of the bath from pH 7.4 to 6.02 led to an immediate increase in conductance and slight reduction in the inward rectification. Further acidification to pH 4.80 led to an additional increase in conductance and a nearly linear current-voltage relation without shifting the reversal potential. The apparent pKa for the conductance change was 5.68 ± 0.08 (n = 4; Fig. 15 B).

Bottom Line: Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot.Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability.The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Oregon Health Sciences University, Portland, OR 97201, USA.

ABSTRACT
The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

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