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Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels.

Tabcharani JA, Linsdell P, Hanrahan JW - J. Gen. Physiol. (1997)

Bottom Line: This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4).The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide.We propose that CFTR displays a "weak field strength" anion selectivity sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, McGill University, Montréal, Québec Canada H3G 1Y6.

ABSTRACT
Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. The current-voltage relationship became linear when patches were excised into symmetrical, -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22 degrees C to 10.9 pS at 37 degrees C. The conductance at 22 degrees C was approximately 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl activity was hyperbolic and well fitted by a Michaelis-Menten-type function having a of approximately 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P/P = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P(1.8) > P(1. 3) > P(1.0) > P(0.17), consistent with a "weak field strength" selectivity site. The same sequence was obtained for external halides, although inward F flow was not observed. Iodide currents were protocol dependent and became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4). The switch to low I permeability was enhanced at potentials that favored Cl entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl and I ions may influence I permeation and be responsible for the wide range of P/P ratios that have been reported for the CFTR channel. The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a "weak field strength" anion selectivity sequence.

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Hysteresis of the current–voltage relationship with intracellular iodide solution. (A, left) Traces recorded at different  holding potentials in the sequence are indicated; i.e., starting at  negative potentials where currents would initially be carried by iodide. (right) Traces obtained from the same patch after the switch  to low I− permeability. Note that current amplitudes decreased by  ∼40% at +80 mV, and iodide currents could no longer be measured at negative potentials. (B) Mean current–voltage relationship obtained under biionic conditions with internal iodide (mean  ± SEM, n = 6). Arrows indicate the protocol. Squares represent  initial currents in the Iunbl state, when the mean reversal potential  indicated PI/PCl = 2.1. Inverted triangles show the currents measured after channels switched to low I− permeability (Ibl state). Positive currents, which would be carried by external I− ions, could no  longer be measured under these conditions. The extrapolated reversal potential under these conditions indicated PI << PCl.
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Figure 8: Hysteresis of the current–voltage relationship with intracellular iodide solution. (A, left) Traces recorded at different holding potentials in the sequence are indicated; i.e., starting at negative potentials where currents would initially be carried by iodide. (right) Traces obtained from the same patch after the switch to low I− permeability. Note that current amplitudes decreased by ∼40% at +80 mV, and iodide currents could no longer be measured at negative potentials. (B) Mean current–voltage relationship obtained under biionic conditions with internal iodide (mean ± SEM, n = 6). Arrows indicate the protocol. Squares represent initial currents in the Iunbl state, when the mean reversal potential indicated PI/PCl = 2.1. Inverted triangles show the currents measured after channels switched to low I− permeability (Ibl state). Positive currents, which would be carried by external I− ions, could no longer be measured under these conditions. The extrapolated reversal potential under these conditions indicated PI << PCl.

Mentions: Similar hysteresis and block were observed with I− on the cytoplasmic side (Fig. 8). Iodide currents could be recorded for several minutes with I− in the bath and Cl− in the pipette if patches were excised while holding the membrane at negative potentials. When the potential was increased, the resulting i/V relationship was outwardly rectified and reversed near +16 mV, indicating PI/PCl = 1.8, but transitions abruptly switched from the unblocked (Iunbl) state to the “blocked” (Ibl) state have ∼40% lower conductance. Entering Ibl coincided with a negative shift in the (extrapolated) reversal potential to at least −20 mV, consistent with a large decrease in relative iodide permeability to PI/PCl ± 0.4 (n = 6–10), similar to that reported in many previous studies of macroscopic I− conductance (e.g., Anderson et al., 1991; Bell and Quinton, 1992). Each point in Fig. 8 is the mean of 3–10 observations; however, points were collected from at least 20 patches because the unblocked state was transient and not all i/V points could be obtained from each patch. It was not possible to record iodide currents after the switch between Ibl and Iunbl had occurred; however, the number of CFTR channels observed before and after were always similar. When channels had been allowed to enter the Ibl state and the bath solution containing NaI, MgATP, and PKA was completely replaced by NaCl, transitions initially disappeared, consistent with the known dependence of CFTR gating on ATP. Restoring MgATP and PKA to the NaCl solution reactivated Iunbl transitions, allowing Cl− currents to be recorded at both positive and negative holding potentials.


Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels.

Tabcharani JA, Linsdell P, Hanrahan JW - J. Gen. Physiol. (1997)

Hysteresis of the current–voltage relationship with intracellular iodide solution. (A, left) Traces recorded at different  holding potentials in the sequence are indicated; i.e., starting at  negative potentials where currents would initially be carried by iodide. (right) Traces obtained from the same patch after the switch  to low I− permeability. Note that current amplitudes decreased by  ∼40% at +80 mV, and iodide currents could no longer be measured at negative potentials. (B) Mean current–voltage relationship obtained under biionic conditions with internal iodide (mean  ± SEM, n = 6). Arrows indicate the protocol. Squares represent  initial currents in the Iunbl state, when the mean reversal potential  indicated PI/PCl = 2.1. Inverted triangles show the currents measured after channels switched to low I− permeability (Ibl state). Positive currents, which would be carried by external I− ions, could no  longer be measured under these conditions. The extrapolated reversal potential under these conditions indicated PI << PCl.
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Related In: Results  -  Collection

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Figure 8: Hysteresis of the current–voltage relationship with intracellular iodide solution. (A, left) Traces recorded at different holding potentials in the sequence are indicated; i.e., starting at negative potentials where currents would initially be carried by iodide. (right) Traces obtained from the same patch after the switch to low I− permeability. Note that current amplitudes decreased by ∼40% at +80 mV, and iodide currents could no longer be measured at negative potentials. (B) Mean current–voltage relationship obtained under biionic conditions with internal iodide (mean ± SEM, n = 6). Arrows indicate the protocol. Squares represent initial currents in the Iunbl state, when the mean reversal potential indicated PI/PCl = 2.1. Inverted triangles show the currents measured after channels switched to low I− permeability (Ibl state). Positive currents, which would be carried by external I− ions, could no longer be measured under these conditions. The extrapolated reversal potential under these conditions indicated PI << PCl.
Mentions: Similar hysteresis and block were observed with I− on the cytoplasmic side (Fig. 8). Iodide currents could be recorded for several minutes with I− in the bath and Cl− in the pipette if patches were excised while holding the membrane at negative potentials. When the potential was increased, the resulting i/V relationship was outwardly rectified and reversed near +16 mV, indicating PI/PCl = 1.8, but transitions abruptly switched from the unblocked (Iunbl) state to the “blocked” (Ibl) state have ∼40% lower conductance. Entering Ibl coincided with a negative shift in the (extrapolated) reversal potential to at least −20 mV, consistent with a large decrease in relative iodide permeability to PI/PCl ± 0.4 (n = 6–10), similar to that reported in many previous studies of macroscopic I− conductance (e.g., Anderson et al., 1991; Bell and Quinton, 1992). Each point in Fig. 8 is the mean of 3–10 observations; however, points were collected from at least 20 patches because the unblocked state was transient and not all i/V points could be obtained from each patch. It was not possible to record iodide currents after the switch between Ibl and Iunbl had occurred; however, the number of CFTR channels observed before and after were always similar. When channels had been allowed to enter the Ibl state and the bath solution containing NaI, MgATP, and PKA was completely replaced by NaCl, transitions initially disappeared, consistent with the known dependence of CFTR gating on ATP. Restoring MgATP and PKA to the NaCl solution reactivated Iunbl transitions, allowing Cl− currents to be recorded at both positive and negative holding potentials.

Bottom Line: This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4).The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide.We propose that CFTR displays a "weak field strength" anion selectivity sequence.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, McGill University, Montréal, Québec Canada H3G 1Y6.

ABSTRACT
Permeation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels by halide ions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. In cell-attached patches with a high Cl pipette solution, the CFTR channel displayed outwardly rectifying currents and had a conductance near the membrane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. The current-voltage relationship became linear when patches were excised into symmetrical, -tris(hydroxymethyl)methyl-2-aminomethane sulfonate (TES)-buffered solutions. Under these conditions, conductance increased from 7.0 pS at 22 degrees C to 10.9 pS at 37 degrees C. The conductance at 22 degrees C was approximately 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent block by pH buffers. The relationship between conductance and Cl activity was hyperbolic and well fitted by a Michaelis-Menten-type function having a of approximately 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indicated high anion selectivity (P/P = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic side to various test anions indicated P(1.8) > P(1. 3) > P(1.0) > P(0.17), consistent with a "weak field strength" selectivity site. The same sequence was obtained for external halides, although inward F flow was not observed. Iodide currents were protocol dependent and became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly reduced I/Cl permeability ratio (P/P< 0.4). The switch to low I permeability was enhanced at potentials that favored Cl entry into the pore and was not observed in the R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. Interactions between Cl and I ions may influence I permeation and be responsible for the wide range of P/P ratios that have been reported for the CFTR channel. The low P/P ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR displays a "weak field strength" anion selectivity sequence.

Show MeSH
Related in: MedlinePlus