ABCMdb

PMID: 14610019

Gong X, Linsdell P
Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore.
J Gen Physiol. 2003 Dec;122(6):673-87. Epub 2003 Nov 10., [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)4 2- ions. Login to comment 5 ABCC7 p.Phe337Ala Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Login to comment 6 ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr Current in the presence of Pt(NO2)4 2- increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)4 2- ions to the extracellular solution. Login to comment 8 ABCC7 p.Phe337Ala Relief of block in F337A by Pt(NO2)4 2- permeation was only observed for blocker concentrations above 300 ␮M; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl- current. Login to comment 9 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)4 2in F337A, an apparent manifestation of an anomalous mole fraction effect. Login to comment 10 ABCC7 p.Phe337Ala We suggest that the F337A mutation allows intracellular Pt(NO2)4 2to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)4 2- ions within the pore promotes their permeation to the extracellular solution. Login to comment 98 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys Block of R334C and S341A appeared somewhat weaker than for wild-type CFTR, whereas K335A and T338A showed a similar degree of block as wild-type (Fig. 5, A-C). Login to comment 100 ABCC7 p.Phe337Ala In contrast, block of F337A was poorly described by the Woodhull model (Fig. 5 B), with block of this mutant appearing to be very much more voltage dependent at negative voltages than at positive voltages (Fig. 5 B). Login to comment 101 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala Although estimation of the blocking effects of Pt(NO2)4 2at 0 mV membrane potential suggested a slight but significant weakening of block in F337A compared with wild-type (Fig. 5 C), direct comparison of the blocking effects of 300 ␮M Pt(NO2)4 2- on wild-type and F337A (Fig. 5 D) suggests that while block is weakened in this mutant at depolarized voltages, the block is actually stronger in F337A than in wild-type at strongly hyperpolarized voltages. Login to comment 102 ABCC7 p.Phe337Tyr Neither of these effects were observed in another F337 mutant, F337Y, which was blocked in an apparently similar manner as wild-type (Fig. 5, C and E). Login to comment 103 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala The Interaction between Pt(NO2)4 and F337A-CFTR Compared with the unremarkable block of wild-type CFTR by intracellular Pt(NO2)4 2- (Figs. 1-3), block of F337A-CFTR appears complex. Login to comment 105 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr However, when we investigated the block at the most negative voltages that we were able to keep membrane patches (-150 mV) with a low extracellular Cl-concentration (4 mM), we noticed an anomalous voltage-dependent increase in Pt(NO2)4 2--blocked current in F337A but not in wild-type, F337Y or T338A (Fig. 6). Login to comment 106 ABCC7 p.Thr338Ala ABCC7 p.Phe337Tyr Under these conditions, the strength of Pt(NO2)4 2- block in wild-type, F337Y, and T338A increases with increasingly negative voltages, eventually leading to a negative slope of the current-voltage relationship in the presence of blocker (Fig. 6 B). Login to comment 107 ABCC7 p.Phe337Ala However, in F337A, the current in the presence of blocker increases again at voltages more negative than around -80 mV, suggesting that as the membrane potential is made very negative blocking ions are swept from the pore and Cl- is able more easily to permeate. Login to comment 116 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr Thus, at very negative voltages, Pt(NO2)4 2- ions can escape from the F337A channel pore, but apparently not from the pore of wild-type, F337Y or T338A, by passing through the channel and into the extracellular solution-a process previously termed "punchthrough" (Nimigean and Miller, 2002). Login to comment 117 ABCC7 p.Phe337Ala Interestingly, Pt(NO2)4 2- punchthrough in F337A was observed at low (Fig. 6) but not high extracellular Cl- concentrations (Fig. 7), suggesting that extracellular Cl- ions can prevent Pt(NO2)4 2- from passing through this mutant channel. Login to comment 120 ABCC7 p.Phe337Ala Concentration-inhibition experiments with low extracellular Cl- concentrations confirmed the multiple apparent effects of the F337A mutation on the apparent affinity of Pt(NO2)4 2- block (Fig. 8). Login to comment 121 ABCC7 p.Phe337Ala At relatively depolarized voltages (e.g., 0 mV; Fig. 8 C), Pt(NO2)4 2- blocked wild-type more strongly than F337A (i.e., the concentration-inhibition curve for wild-type lies to the left); whereas at hyperpolarized voltages (e.g., -130 mV, Fig. 8 D), the mutant is more potently inhibited. Login to comment 122 ABCC7 p.Phe337Ala However, these experiments also illustrate that the punchthrough mechanism that relieves block of F337A but not wild-type at strongly hyperpolarized voltages is dependent not only on voltage but also on the blocker concentration. Login to comment 128 ABCC7 p.Phe337Ala Confirming that Pt(NO2)4 2- can itself relieve Pt(NO2)4 2- block of F337A-CFTR, increasing the concentration of blocker from 100 to 300 ␮M during an individual experiment reduced current amplitude over most of the voltage range, but anomalously increased current amplitude below about -100 mV (Fig. 9, C-E). Login to comment 131 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala This suggests that the ability of Pt(NO2)4 2to permeate through the F337A channel pore is dependent on its own concentration. Login to comment 132 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala While we have not attempted to estimate the "permeability" of Pt(NO2)4 2in F337A-CFTR, we note Figure 4. Login to comment 139 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala To ensure that this did not, in fact, reflect time-dependent changes in F337A current amplitude, Pt(NO2)4 2- block of F337A was also studied using a voltage-step protocol (Fig. 10). Login to comment 140 ABCC7 p.Phe337Ala F337A-CFTR currents were practically time-independent in the absence and presence of Pt(NO2)4 2- (Fig. 10 A). Login to comment 145 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys (A) Example macroscopic currents carried by the CFTR mutants R334C, K335A, F337A, T338A, and S341A before (Control) and after addition of 300 ␮M Pt(NO2)4 2to the intracellular solution. Login to comment 147 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys Each plot has been fitted by Eq. 2; this provides a good fit of R334C (Kd(0) ϭ 2080 ␮M, z␦ ϭ -0.174), K335A (Kd(0) ϭ 418 ␮M, z␦ ϭ -0.317), T338A (Kd(0) ϭ 626 ␮M, z␦ ϭ -0.351) and S341A (Kd(0) ϭ 1362 ␮M, z␦ ϭ -0.249), but a poor fit of F337A. Login to comment 148 ABCC7 p.Phe337Ala (C) Mean Kd(0) estimated from fits such as those shown in B, except for F337A where Kd(0) was calculated from the fractional current remaining (I/I0) at 0 mV (estimated by fitting a polynomial function) according to the equation Kd(0) ϭ (I (300 ␮M))/(I0 - I). Login to comment 150 ABCC7 p.Phe337Ala (D) Comparison of the mean blocking effect of 300 ␮M intracellular Pt(NO2)4 2- on wild-type (᭺; fitted by Eq. 2 as described in Fig. 2) and F337A (᭹; fitted by a third order polynomial function of no theoretical significance). Login to comment 151 ABCC7 p.Phe337Tyr (E) Comparison of the mean blocking effect of 300 ␮M intracellular Pt(NO2)4 2- on wild-type (᭺; fitted as in D) and F337Y (᭹; fitted by Eq. 2 with Kd(0) ϭ 582 ␮M and z␦ ϭ -0.318). Login to comment 154 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala Punchthrough of Pt(NO2)4 2in F337A was blocked by extracellular Cl- ions (Fig. 7). Login to comment 157 ABCC7 p.Phe337Ala At very negative voltages, however, Pt(NO2)4 2- block of F337A is anomalously strengthened by high extracellular Cl- concentrations (Fig. 12). Login to comment 162 ABCC7 p.Phe337Ala Apparent Pt(NO2)4 2- unblock by permeation in F337A. Login to comment 173 ABCC7 p.Phe337Ala Pt(NO2)4 2- punchthrough in F337A is prevented by extracellular permeant anions. Login to comment 174 ABCC7 p.Phe337Ala (A) Example macroscopic currents carried by F337A-CFTR before (Control) and after addition of 1 mM Pt(NO2)4 2to the intracellular solution, with 150 mM chloride, nitrate or perchlorate present in the extracellular solution. Login to comment 178 ABCC7 p.Phe337Ala Comparison of the blocking effects of intracellular Pt(NO2)4 2- on wild-type and F337A-CFTR at low extracellular Cl-concentration. Login to comment 179 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala (A and B) Mean fraction of control current remaining following addition of 3 ␮M (᭹), 10 ␮M (᭺), 30 ␮M (᭢), 100 ␮M (᭞), 300 ␮M (᭿), or 1 mM (ٗ) Pt(NO2)4 2to the intracellular solution, for wild-type (A) and F337A (B). Login to comment 180 ABCC7 p.Phe337Ala (C and D) Comparison of the concentration dependence of block in wild-type (᭹) and F337A (᭺) at two different membrane potentials: 0 mV (C) and -130 mV (D). Login to comment 184 ABCC7 p.Phe337Ala Our interest in this substance stems from the consequences of a mutation within the pore (F337A) that apparently turns the channel from being Pt(NO2)4 2- impermeable to Pt(NO2)4 2- permeable (Fig. 6) and destroys the apparent simplicity of blocking effect seen in wild-type. Login to comment 186 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala However, punchthrough of Pt(NO2)4 2at negative voltages suggests that this anion is capable of passing through the pore of F337A-CFTR (Figs. 6, 7, and 9-11). Login to comment 187 ABCC7 p.Phe337Ala As described by Nimigean and Miller (2002), the punchthrough phenomenon may be able to reveal very low levels of permeability inaccessible by other experimental means, and punchthrough of Pt(NO2)4 2-was only observed under highly specific conditions (in F337A only, at voltages more negative than approximately -80 mV, low extracellular permeant anion concentration, and Pt(NO2)4 2- concentrations of at least 300 ␮M). Login to comment 188 ABCC7 p.Phe337Ala Nevertheless, the results shown in Fig. 6 suggest that the F337A mutation confers Pt(NO2)4 2- permeability on the pore. Login to comment 189 ABCC7 p.Phe337Ser ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr Previously, we showed that the mutations F337A and F337S, but not F337Y, disrupted the ability of the CFTR channel pore to select between permeant anions on the basis of free energy of hydration (Linsdell et al., 2000) and suggested that F337 contributes to a lyotropic anion "selectivity filter." Login to comment 190 ABCC7 p.Thr338Ala In contrast, mutations of the adjacent TM6 residue (T338), including T338A, altered the selectivity between different lyotro- Figure 9. Login to comment 201 ABCC7 p.Thr338Ala ABCC7 p.Phe337Ala The slight Pt(NO2)4 2- permeability of F337A therefore suggests that this divalent anion might normally be prevented from passing through the pore for similar reasons that limit the permeability of kosmotropic anions like F-. In contrast, the T338A mutation appears to enhance unblock by permeation of the lyotropic Au(CN)2 - ion (Gong and Linsdell, 2003b). Login to comment 203 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala In addition to allowing Pt(NO2)4 2- permeability, the F337A mutation has a complex effect on the apparent affinity of Pt(NO2)4 2- block (Figs. 5 D and 8, B and D): block appears weaker than for wild-type at positive voltages yet stronger than in wild-type at negative voltages (and then weakens again in F337A due to punchthrough; see below). Login to comment 204 ABCC7 p.Phe337Ala The block observed in F337A is poorly fitted by conventional models that assume a single binding site (Fig. 5 B). Login to comment 205 ABCC7 p.Phe337Ala We suggest that this reflects binding to more than one site in the F337A-CFTR pore; a low affinity site that is accessible at all voltages, and a higher affinity site that is increasingly accessed at more negative voltages. Login to comment 206 ABCC7 p.Phe337Ala The existence of more than one Pt(NO2)4 2-binding site in the F337A pore is also supported by the apparent anomalous mole fraction dependence of Pt(NO2)4 2- permeability (Fig. 9). Login to comment 207 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala ABCC7 p.Phe337Tyr Since this complex blocking behavior is observed in F337A but not in wild-type or F337Y, we suggest that by allowing Pt(NO2)4 2to permeate through the pore, the F337A mutant also allows this blocker to reach a binding site which is normally inaccessible or much less easily accessed. Login to comment 209 ABCC7 p.Phe337Ala A simple model of Pt(NO2)4 2- movement in the F337A pore is shown in Fig. 13. Login to comment 210 ABCC7 p.Phe337Ala Even in this mutant, Pt(NO2)4 2- unblock by permeation only occurs under extreme conditions (strongly hyperpolarized voltages, low extracellular Cl- concentrations, and high Pt(NO2)4 2- concentration; Fig. 6), such that it appears that the blocker normally exits the F337A pore back into the intracellular solution. Login to comment 212 ABCC7 p.Phe337Ala Pt(NO2)4 2- block of F337A investigated using a voltage-step protocol. Login to comment 213 ABCC7 p.Phe337Ala (A) Example F337A-CFTR currents in an inside-out patch, recorded before current activation (Control), after full current activation with PKA and PPi, and following sequential addition of Pt(NO2)4 2to final concentrations of 100 and 300 ␮M. Login to comment 221 ABCC7 p.Phe337Ala overcome in F337A than in wild-type, and a second barrier external to the outermost Pt(NO2)4 2-binding site depicted in Fig. 13. Login to comment 223 ABCC7 p.Phe337Ala ABCC7 p.Phe337Ala With the addition of a second barrier to Pt(NO2)4 2- movement in the pore (Fig. 13), our model appears able to explain the complex interaction between Pt(NO2)4 2and F337A-CFTR. Login to comment 227 ABCC7 p.Phe337Ala At low concentrations of Pt(NO2)4 2-, the blocker returns from the high affinity site in F337A to the intracellular solution (Fig. 13 B). Login to comment 230 ABCC7 p.Phe337Ala Mechanistically, we suggest that at concentrations Ͼ300 ␮M, the F337A pore begins to show multiple occupancy by Pt(NO2)4 2- ions, and that repulsion between simultaneously bound ions is capable of expelling ions bound to the "outer" site into the extracellular solution, relieving the high-affinity block (Fig. 13 C). Login to comment 232 ABCC7 p.Phe337Ala Timecourse of Pt(NO2)4 2- block of F337A investigated using a voltage-step protocol. Login to comment 240 ABCC7 p.Phe337Ala Complex effect of extracellular Cl-concentration on block of F337A-CFTR by 300 ␮M Pt(NO2)4 2-. Login to comment 246 ABCC7 p.Phe337Ala The present results suggest that multiple Pt(NO2)4 2- ions can bind simultaneously within the F337A-CFTR pore (and perhaps also wild-type CFTR), and also that Pt(NO2)4 2-binding may be able to occur concurrently with binding of extracellular Cl- or NO3 - ions. Login to comment 250 ABCC7 p.Phe337Ala Our results suggest that, by removing a barrier to Pt(NO2)4 2- movement in the pore, the F337A mutation allows this anion to access a relatively high affinity binding site and simultaneously exposes it to multiion pore effects that destabilize its binding at high concentrations. Login to comment 275 ABCC7 p.Phe337Ala A pictorial model of Pt(NO2)4 2- block in F337A-CFTR. Login to comment