ABCMdb

PMID: 10866956

Zhang ZR, McDonough SI, McCarty NA
Interaction between permeation and gating in a putative pore domain mutant in the cystic fibrosis transmembrane conductance regulator.
Biophys J. 2000 Jul;79(1):298-313., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC7 p.Ser1118Phe Here we report a serine-to-phenylalanine mutation (S1118F) in the 11th transmembrane domain that confers voltage-dependent, single-exponential current relaxations and moderate inward rectification of the macroscopic currents upon expression in Xenopus oocytes. Login to comment 3 ABCC7 p.Ser1118Phe At steady state, the S1118F-CFTR single-channel conductance rectifies, corresponding to the whole-cell rectification. Login to comment 5 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe S1118F-CFTR currents are blocked in a voltage-dependent manner by diphenylamine-2-carboxylate (DPC); the affinity of S1118F-CFTR for DPC is similar to that of the wild-type channel, but blockade exhibits moderately reduced voltage dependence. Login to comment 22 ABCC7 p.Ser1118Phe Here we report a serine-to-phenylalanine mutation (S1118F) in TM11 that affects both permeation and gating. Login to comment 23 ABCC7 p.Ser1118Phe Most strikingly, the macroscopic S1118F-CFTR currents display voltage-dependent current relaxations; these relaxations are modulated by permeating anions. Login to comment 33 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala ABCC7 p.Phe1111Ser Preparation of oocytes and cRNA CFTR was subcloned into the pALTER vector (Promega), and the S1118F, S1118A, and S1118F/F1111S mutations were made using the Promega Altered Sites protocol (McDonough et al., 1994). Login to comment 80 ABCC7 p.Ser1118Phe Unexpectedly, the most obvious consequence of the mutation of S1118F-CFTR is the appearance of relaxations in current density upon stepping to a new membrane potential. Login to comment 85 ABCC7 p.Ser1118Phe However, S1118F-CFTR displays voltage-dependent current relaxations in response to protocols applying either depolarizing or hyperpolarizing prepotentials. Login to comment 87 ABCC7 p.Ser1118Phe These voltage-jump relaxations lead to a larger conductance for jumps to more negative potentials and to a smaller conductance for jumps in the FIGURE 1 Mutation S1118F-CFTR results in current relaxations. Login to comment 93 ABCC7 p.Ser1118Phe (B and C) Families of currents measured in oocytes expressing either wild-type or S1118F-CFTR channels, respectively. Login to comment 104 ABCC7 p.Ser1118Phe Note that S1118F-CFTR also displays tail currents (Figs. 1, 2, 6, and 7), which have not been described for WT or any mutant of CFTR studied thus far. Login to comment 116 ABCC7 p.Ser1118Phe In WT-CFTR, TM11 contains four phenylalanine residues (Fig. 1 A); mutation of S1118F adds a fifth. Login to comment 117 ABCC7 p.Ser1118Phe If this domain is ॷ-helical, as predicted from hydropathy analysis (Riordan et al., 1989), mutation S1118F would then place three phenylalanine residues in close mutual proximity (at positions 1107, 1111, and 1118) and on approximately the same face of the helix. Login to comment 118 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Phe1111Ser To determine whether this imposed pattern was the source of relaxations in S1118F-CFTR, due to introduction of another bulky residue, the double mutation S1118F/F1111S was constructed. Login to comment 119 ABCC7 p.Ser1118Phe These channels displayed relaxations indistinguishable from those of the single FIGURE 2 Relaxations induced in S1118F-CFTR currents were fit to single-exponential decay functions. Login to comment 125 ABCC7 p.Ser1118Ala These relaxations are not seen in S1118A channels (not shown). Login to comment 126 ABCC7 p.Ser1118Phe mutant S1118F-CFTR (not shown). Login to comment 128 ABCC7 p.Ser1118Ala In addition, mutation of S1118 to alanine (S1118A-CFTR) did not cause relaxations. Login to comment 132 ABCC7 p.Ser1118Phe Gating behavior of single CFTR channels from oocytes expressing WT or S1118F-CFTR was studied in excised patch mode. Login to comment 139 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe Under these conditions, burst duration was 1392 afe; 77 ms for WT-CFTR (n afd; 441 bursts) and 102 afe; 8 ms for S1118F- FIGURE 3 The current-voltage relations for S1118F-CFTR channel shift during the voltage-jump relaxations. Login to comment 152 ABCC7 p.Ser1118Phe Note that S1118F-CFTR channels exhibit much briefer openings than does WT. Login to comment 153 ABCC7 p.Ser1118Phe Currents were not observed in phosphorylated WT or S1118F-CFTR channels bathed in solutions lacking ATP (not shown). Login to comment 155 ABCC7 p.Ser1118Phe Hence the mean burst duration in S1118F-CFTR is reduced to only 7% that of WT channels. Login to comment 166 ABCC7 p.Ser1118Phe We tested whether S1118F-CFTR exhibited alterations in the interaction with DPC, which would be consistent with a pore-lining position for this serine. Login to comment 167 ABCC7 p.Ser1118Phe Fig. 6 shows background-subtracted S1118F-CFTR currents for a representative cell before (Fig. 6 A) and several minutes after (Fig. 6 B) bath application of 200 òe;M DPC. Login to comment 171 ABCC7 p.Ser1118Phe At afa;100 mV, DPC blocks S1118F-CFTR and WT channels with roughly the same efficacy: the apparent KD (at afa;100 mV) was, respectively, 266 afe; 13 òe;M and 276 afe; 14 òe;M (mean afe; SE, n afd; 6 and 15; data for WT are from McDonough et al., 1994). Login to comment 174 ABCC7 p.Ser1118Phe The voltage dependence of the S1118F-CFTR block is significantly less steep than that of the wild type; the apparent binding distance is òa; afd; 0.27 afe; 0.01 (mean afe; SE, n afd; 6) for the mutant versus 0.41 afe; 0.03 for WT channels (McDonough et al., 1994). Login to comment 175 ABCC7 p.Ser1118Phe S1118F-CFTR exhibits altered permeation characteristics S1118, according to our alignment, occupies a position in TM11 that is homologous to that of T338 in TM6. Login to comment 179 ABCC7 p.Ser1118Phe Because oocytes expressing S1118F-CFTR show voltage-jump relaxations, it was not appropriate to use the more common approach of applying voltage-ramp protocols to generate selectivity data. Login to comment 180 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala Instead, currents in oocytes expressing WT-, S1118F-, or S1118A-CFTR channels were elicited by stepping for 75 ms from the holding potential of afa;30 mV to a series of test potentials between afa;140 and af9;80 mV in af9;20 mV increments. Login to comment 182 ABCC7 p.Ser1118Phe If there are two open states that vary in the voltage dependence of microscopic kinetics, currents measured early in the relaxation and at later times likely represent different mixtures of FIGURE 5 S1118F-CFTR channels show rectification of single-channel conductance. Login to comment 189 ABCC7 p.Ser1118Phe Hence currents at each potential were averaged over two time periods: either the first FIGURE 6 Mutation S1118F has mild effects on blockade by DPC. Login to comment 204 ABCC7 p.Ser1118Phe (B) Currents in S1118F-CFTR-expressing oocytes, showing pronounced relaxations at depolarizing test potentials. Login to comment 206 ABCC7 p.Ser1118Ala S1118A-CFTR channels did not indicate time dependence with this protocol (not shown). Login to comment 207 ABCC7 p.Ser1118Phe Note the prominent tail currents in S1118F-CFTR but not in WT. Login to comment 220 ABCC7 p.Arg347Asp This was initially attributed to interaction of this anion with R347 at the cytoplasmic end of TM6 in CFTR, based upon the loss of iodide block in R347D-CFTR. Login to comment 235 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala Data are shown for both instantaneous currents and steady-state currents for WT and S1118F-CFTR (Fig. 8, A and B) or S1118A-CFTR (Fig. 8, C and D). Login to comment 236 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala In Clafa; -containing solutions, before anion substitutions were made, there was a significant difference (p afd; 0.047) between instantaneous and steady-state reversal potentials in S1118F-CFTR (Table 2) compared to the WT (Table 1), but not in S1118A-CFTR. Login to comment 237 ABCC7 p.Ser1118Phe This suggests that S1118F-CFTR channels may be less Clafa; -selective than are WT channels. Login to comment 238 ABCC7 p.Ser1118Phe Comparing the selectivity pattern in S1118F-CFTR with that of WT, without respect to time dependence, showed significant changes for both relative permeabilities and relative conductances (compare Tables 1 and 2). Login to comment 239 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala The ability of the large anions (acetate, gluconate, glutamate, and isethionate) to gain access to the pore is altered by mutations S1118A and S1118F, as indicated by mild to significant changes in relative permeabilities for these anions compared to that in WT channels. Login to comment 242 ABCC7 p.Ser1118Ala S1118A-CFTR exhibited reduced relative permeabilities for all of the large anions and for perchlorate. Login to comment 246 ABCC7 p.Ser1118Phe Accordingly, GX/GCl in S1118F-CFTR showed dramatic changes for seven of the nine test anions (Fig. 8 B). Login to comment 247 ABCC7 p.Ser1118Phe Relative conductances for the large anions were altered substantially in the S1118F-CFTR. Login to comment 249 ABCC7 p.Ser1118Phe S1118F-CFTR also exhibited less block by iodide and thiocyanate, consistent with disruption of the high-affinity binding of these anions in the WT pore. Login to comment 250 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala Relative conductances were generally less affected in S1118A-CFTR than in S1118F-CFTR. Login to comment 251 ABCC7 p.Ser1118Ala In contrast, mutation S1118A had the greatest effects on PX/PCl relationships, particularly for the large polyatomic anions (Fig. 8, C and D). Login to comment 252 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala FIGURE 8 Selectivity data for WT CFTR as compared to S1118F-CFTR (A and B) and S1118A-CFTR (C and D). Login to comment 260 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala &#a7; p b0d; 0.01 for S1118F-CFTR or p afd; 0.02 for S1118A-CFTR. Login to comment 263 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala Nor does relative permeability for S1118F-CFTR (Fig. 8 A) or S1118A-CFTR (Fig. 8 C) show any time dependence. Login to comment 264 ABCC7 p.Ser1118Phe However, relative conductances for the largest anions studied (gluconate and glutamate) decreased in S1118F-CFTR during the 75-ms voltage jump (Fig. 8 B;&#a7; indicates p b0d; 0.01). Login to comment 265 ABCC7 p.Ser1118Phe Because these effects in S1118F-CFTR were only found for the largest anions tested, we propose that this result signifies a time-dependent change in the diameter of the narrowest part of the pore. We would expect this difference to be magnified in experiments in which Clafa; was replaced entirely with glutamate or gluconate (Khakh and Lester, 1999). Login to comment 267 ABCC7 p.Ser1118Ala As shown in Fig. 8 D, there was also a statistically significant time dependence of relative conductance to perchlorate for S1118A-CFTR (p afd; 0.02). Login to comment 269 ABCC7 p.Ser1118Phe Fig. 9 shows the current-voltage relations in the presence of chloride and glutamate for one representative experiment in S1118F-CFTR. Login to comment 274 ABCC7 p.Ser1118Phe Based upon the following observations, we reasoned that there may be a structural connection between the changes in permeation properties of S1118F-CFTR and the process underlying the voltage jump relaxations: 1) The change in permeation properties after a voltage jump (Figs. 1 and 9) is consistent with the steady-state kinetics of single channels (Fig. 5). Login to comment 278 ABCC7 p.Ser1118Phe To address this issue, we asked whether there is any interaction between permeation and gating in S1118F-CFTR channels studied under whole-cell conditions. Login to comment 280 ABCC7 p.Ser1118Phe Current-voltage relations in chloride and glutamate for instantaneous currents (--) and steady-state currents (- - -) in S1118F-CFTR were adjusted on the voltage axis so that their reversal potentials were set to zero. Login to comment 284 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala ABCC7 p.Ser1118Ala TABLE 2 Selectivity in S1118F-CFTR and S1118A-CFTR Ion S1118F S1118A Erev (mV) PX/PCl GX/GCl Erev (mV) PX/PCl GX/GCl SCN afa;40.97 afe; 1.20* 1.84 afe; 0.08* 0.57 afe; 0.02* afa;56.44 afe; 0.93 2.59 afe; 0.09 0.20 afe; 0.01 NO3 afa;32.83 afe; 1.69 1.30 afe; 0.06 0.95 afe; 0.02 afa;37.35 afe; 0.87 1.21 afe; 0.04 0.88 afe; 0.01 Br afa;28.28 afe; 1.32 1.07 afe; 0.03 0.88 afe; 0.03* afa;34.53 afe; 0.85 1.12 afe; 0.02 0.77 afe; 0.01 Cl afa;26.14 afe; 1.59* 1.0 1.0 afa;30.63 afe; 0.64 1.0 1.0 I afa;8.46 afe; 1.14 0.52 afe; 0.03* 0.38 afe; 0.03* afa;10.35 afe; 1.26 0.39 afe; 0.01 0.27 afe; 0.05 Acetate 39.64 afe; 1.77* 0.05 afe; 0.01* 0.12 afe; 0.01* 23.24 afe; 1.83* 0.09 afe; 0.01* 0.50 afe; 0.01* Glutamate 23.93 afe; 3.61* 0.16 afe; 0.01* 0.23 afe; 0.01* 19.59 afe; 1.07* 0.09 afe; 0.01* 0.49 afe; 0.01* 0.32 afe; 0.01I * Isethionate 20.24 afe; 3.62* 0.14 afe; 0.03 0.25 afe; 0.02* 23.71 afe; 0.99* 0.09 afe; 0.01* 0.48 afe; 0.01 ClO4 afa;6.42 afe; 1.57* 0.42 afe; 0.02* 0.18 afe; 0.01 27.29 afe; 1.36* 0.06 afe; 0.01* 0.12 afe; 0.01* 0.14 afe; 0.01I * Gluconate 28.26 afe; 3.29* 0.12 afe; 0.02 0.20 afe; 0.01* 20.03 afe; 1.04* 0.10 afe; 0.01* 0.50 afe; 0.01* 0.27 afe; 0.01I * Ions are listed in the same order as in Table 1. Login to comment 288 ABCC7 p.Ser1118Phe WT or S1118F-CFTR channels were studied using the same voltage-clamp protocol as the one used for the data shown in Fig. 7. Login to comment 295 ABCC7 p.Ser1118Phe S1118F-CFTR currents showed a relaxation as expected from previous experiments (Figs. 1, 2, and 7). Login to comment 298 ABCC7 p.Ser1118Phe Because SCNafa; blocks mutant channels as it does WT channels (Table 2), we would expect the time constant for S1118F-CFTR channels in the presence of SCNafa; to be reduced as it is for WT channels. Login to comment 299 ABCC7 p.Ser1118Phe In contrast, the relaxations in S1118F-CFTR were slowed by this less permeant anion such that the process underlying the relaxations was not complete even by the end of the 75-ms voltage jump. Login to comment 304 ABCC7 p.Ser1118Phe In summary, the lengthening of the gating process at positive potentials in S1118F-CFTR follows the relative conductance sequence. Login to comment 306 ABCC7 p.Ser1118Phe This implies a distinct connection between permeation and gating in S1118F-CFTR. Login to comment 314 ABCC7 p.Ser1118Phe (C) Time constants for the relaxations in WT and S1118F-CFTR channels were calculated by fitting the decay to an exponential function. Login to comment 315 ABCC7 p.Ser1118Phe Shown are the time constants (঄) for S1118F-CFTR currents recorded in bath solution containing Clafa; , Brafa; , or SCNafa; . Login to comment 316 ABCC7 p.Ser1118Phe surements in S1118F-CFTR show modest current relaxations, on the order of 30%, during voltage jumps between afa;140 and af9;80 mV. Login to comment 321 ABCC7 p.Arg347Asp Anomalous mole fraction effects (Tabcharani et al., 1993) and protocol-dependent block by iodide (Tabcharani et al., 1997) are lost in R347D-CFTR; this may be due to a severe disruption in secondary structure by the loss of a salt bridge between R347 and D924 (Cotten and Welsh, 1999). Login to comment 334 ABCC7 p.Thr1134Phe In particular, mutation T1134F in TM12 lowered single-channel conductance to b03;6 pS and increased DPC affinity without changing the voltage dependence of block (McDonough et al., 1994). Login to comment 335 ABCC7 p.Ser341Ala Mutation S341A in TM6 reduced affinity for DPC fivefold, induced inward rectification, and decreased the single-channel conductance to b03;1 pS (McDonough et al., 1994). Login to comment 338 ABCC7 p.Thr338Ala Mutation T338A altered the voltage dependence of block by DPC without affecting affinity at afa;100 mV (McDonough et al., 1994). Login to comment 343 ABCC7 p.Ser1118Phe ABCC7 p.Thr338Ala With respect to block by DPC, S1118F-CFTR had an effect much like that of T338A-CFTR, wherein affinity at afa;100 mV was not changed significantly but the voltage dependence was reduced. Login to comment 344 ABCC7 p.Ser1118Phe The absolute difference in the apparent KD (at afa;100 mV) for DPC between wild type and S1118F-CFTR, taken by itself, is too small to conclude that residue S1118 lines the channel pore. Login to comment 345 ABCC7 p.Ser1118Ala Similarly, S1118A did not affect block by DPC (McDonough et al., 1994). Login to comment 346 ABCC7 p.Ser1118Phe ABCC7 p.Ser1118Ala However, both S1118A-CFTR and S1118F-CFTR altered the selectivity behavior of the pore, suggesting that this position may contribute to the pore walls. Login to comment 347 ABCC7 p.Ser1118Phe Relative conductances for many of the substitute anions were altered in S1118F-CFTR. Login to comment 348 ABCC7 p.Ser1118Ala In contrast, mutation S1118A exhibited the greatest effects on relative permeabilities, especially for the large anions. Login to comment 353 ABCC7 p.Ser1118Phe Furthermore, S1118F-CFTR also exhibits a reduced single-channel conductance. Login to comment 365 ABCC7 p.Ser1118Phe S1118F-CFTR single channels differ doubly from wild-type channels. Login to comment 366 ABCC7 p.Ser1118Phe First, S1118F-CFTR openings are much briefer than wild-type openings (burst duration is reduced by 93%). Login to comment 367 ABCC7 p.Arg117His ABCC7 p.Ser1118Phe The choppy bursts of S1118F-CFTR closely resemble the bursts of mutant R117H, a mutation that causes mild cystic fibrosis (Sheppard et al., 1993). Login to comment 368 ABCC7 p.Ser1118Phe Second, the single-channel conductance of S1118F-CFTR rectifies inwardly, in agreement with the steady-state rectification of macroscopic currents. Login to comment 369 ABCC7 p.Ser1118Phe The rectification of single-channel steady-state conductance for S1118F-CFTR arises primarily from decreased conductance at positive potentials, because the single-channel conductances for the WT and mutant are nearly equal at afa;100 mV but differ at af9;100 mV. Login to comment 371 ABCC7 p.Ser1118Phe That S1118F-CFTR has more defective conduction properties for positive current at depolarizing potentials (i.e., for inward flux of Clafa; ) suggests that the mutated residue affects the channel at a point nearer the extracellular than the cytoplasmic end of the channel. Login to comment 375 ABCC7 p.Ser1118Phe The shortened openings in S1118F-CFTR may reflect a destabilization of the global protein structure rather than a specific effect on the pore. Login to comment 377 ABCC7 p.Ser1118Ala Similarly, the current relaxations are not due to disruption of a putative interaction between S1118 and another amino acid, because no relaxations were observed for S1118A-CFTR. Login to comment 383 ABCC7 p.Ser1118Phe The single-channel recordings confirm that S1118F-CFTR openings are interrupted to a much greater extent than wild-type openings. Login to comment 389 ABCC7 p.Ser1118Phe Thus there is no reason to suspect that the relaxations are produced by voltage-dependent blocking events, and we tentatively favor the explanation that the S1118F-CFTR has altered gating kinetics. Login to comment 391 ABCC7 p.Ser1118Phe Although an additional state may have been added to the kinetic scheme for S1118F-CFTR, it is also possible that an existing state has been modified. Login to comment 392 ABCC7 p.Ser1118Phe For instance, it will be important to determine if the prominent voltage-dependent fast flicker in WT observed at hyperpolarizing potentials is modified by the S1118F mutation. Login to comment 393 ABCC7 p.Ser1118Phe Possible mechanisms S1118F-CFTR exhibits alterations in permeation properties, as might be expected given the location of this mutation, but also exhibits alterations in single-channel gating. Login to comment 397 ABCC7 p.Ser1118Phe It seems more likely that a unique process occurs for S1118F-CFTR. Login to comment 401 ABCC7 p.Ser1118Phe At the other end of the scale, the S1118F mutation might disrupt the stability of the protein global structure, leading to the observed effects on conduction. Login to comment 405 ABCC7 p.Ser1118Phe The relative conductance of the S1118F-CFTR channel to large anions decreases during the course of the relaxations, which may indicate that either the large anions are more capable of blocking the channels in the steady-state conformation, or that Clafa; permeation becomes blocked by an intrinsic portion of the protein in the steady-state conformation. Login to comment 418 ABCC7 p.Ser1118Phe The S1118F mutation may provide this reporter group in the form of the Phe side chain and/or slow the relaxations to a rate that is resolvable in macroscopic recordings. Login to comment