ABCMdb

PMID: 11741825

Zhang ZR, Zeltwanger S, Smith SS, Dawson DC, McCarty NA
Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR.
Am J Physiol Lung Cell Mol Physiol. 2002 Jan;282(1):L135-45., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Val317Glu ABCC7 p.Val317Glu Am J Physiol Lung Cell Mol Physiol 282: L135-L145, 2002.-A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. Login to comment 10 ABCC7 p.Val317Glu ABCC7 p.Val317Glu In excised patches containing one to a few channels, the time-averaged single-channel current (I)-V relationship (I ϭ N ϫ Po ϫ i, where N is the number of active channels and Po is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the Po of V317E CFTR is voltage dependent. Login to comment 18 ABCC7 p.Val317Glu We studied the impact on channel function of a mutation (V317E) at a site in the extracellular half of the fifth transmembrane domain (TM5). Login to comment 21 ABCC7 p.Val317Glu V317E CFTR macroscopic currents display marked rectification. Login to comment 23 ABCC7 p.Val317Glu Instead, we found that the macroscopic rectification of V317E CFTR was due to a voltage dependence of the open probability (Po) of single channels. Login to comment 29 ABCC7 p.Val317Glu ABCC7 p.Val317Gln cRNA was prepared from a construct carrying the full coding region of CFTR in the pALTER vector (Promega, Madison, WI) for WT CFTR or in pBluescript (Stratagene, La Jolla, CA) for V317E and V317Q CFTR. Login to comment 87 ABCC7 p.Val317Glu Figure 1 shows families of whole cell currents from TEVC experiments in WT and V317E CFTR (Fig. 1, A and B, respectively). Login to comment 93 ABCC7 p.Val317Glu In contrast, V317E CFTR currents exhibited time-dependent behavior at all voltages (Fig. 1B); inward currents relaxed toward zero, while outward currents increased in conductance through time. Login to comment 98 ABCC7 p.Val317Glu V317E CFTR also exhibited modest tail currents (Fig. 1B) not seen in WT CFTR with this voltage protocol. Login to comment 99 ABCC7 p.Val317Glu ER in ND96 bath solution did not differ between WT CFTR (-28.1 Ϯ 0.59 mV; n ϭ 12) and V317E CFTR (-27.1 Ϯ 0.58 mV; n ϭ 10). Login to comment 100 ABCC7 p.Val317Glu Despite the strong change in rectification during the voltage pulse (Fig. 1D), the ER in V317E CFTR was not time dependent. Login to comment 101 ABCC7 p.Val317Glu Hence, selectivity for Clover Naϩ did not appear to differ between WT and the mutant and did not change during the time-dependent behavior of V317E CFTR. Login to comment 103 ABCC7 p.Val317Glu ABCC7 p.Val317Glu Gϩ80/G-80 was 1.67 Ϯ 0.13 for WT CFTR initial and late currents and was 1.52 Ϯ 0.08 for V317E CFTR initial currents (P ϭ 0.004 compared with WT CFTR), and 2.24 Ϯ 0.20 for V317E CFTR late currents (P Ͻ 0.001 compared with WT CFTR). Login to comment 104 ABCC7 p.Val317Glu ABCC7 p.Val317Glu ABCC7 p.Val317Glu The smaller degree of rectification of initial currents in V317E CFTR compared with WT CFTR may reflect reduced susceptibility of V317E CFTR currents to blockage by the cytoplasmic constituent(s) or may reflect an impact of holding potential on the process underlying the rectification (see Mechanism of the voltage dependence of V317E CFTR). Login to comment 108 ABCC7 p.Val317Glu To identify the mechanism responsible for the pronounced outward rectification of V317E CFTR whole cell currents, single-channel studies were performed. Login to comment 113 ABCC7 p.Val317Glu Mutation V317E in putative pore transmembrane domain (TM) 5 results in rectification of cystic fibrosis transmembrane conductance regulator (CFTR) whole cell currents. Login to comment 115 ABCC7 p.Val317Glu B: family of currents for an oocyte expressing V317E CFTR. Login to comment 117 ABCC7 p.Val317Glu Solid lines in C and D only connect the points; dashed lines indicate linear fit of the initial outward currents, indicating that these currents for both WT and V317E CFTR showed no rectification. Login to comment 120 ABCC7 p.Val317Glu ABCC7 p.Val317Glu L137VOLTAGE-SENSITIVE GATING OF V317E CFTR AJP-Lung Cell Mol Physiol • VOL 282 • JANUARY 2002 • www.ajplung.org contrast, the i-V relationship for V317E CFTR was found to exhibit slight voltage dependence, with g ϭ 7.65 Ϯ 0.14 pS at negative potentials and g ϭ 6.34 Ϯ 0.14 pS at positive potentials (n ϭ 7 patches; Fig. 2, C and D). Login to comment 121 ABCC7 p.Val317Glu i Amplitude for V317E CFTR was 0.64 Ϯ 0.02 pA at ϩ100 mV and 0.77 Ϯ 0.03 pA at -100 mV (P Ͻ 0.02). Login to comment 122 ABCC7 p.Val317Glu Thus i at positive potentials for V317E CFTR exhibited only 87% of the conductance of WT CFTR. Login to comment 124 ABCC7 p.Val317Gln To clarify the mechanism leading to reduced g, we studied V317Q. Login to comment 126 ABCC7 p.Val317Gln The i-V relationship of V317Q CFTR was found to have slight voltage dependence, with g ϭ 7.28 Ϯ 0.16 pS at negative potentials and g ϭ 5.91 Ϯ 0.06 pS at positive potentials (n ϭ 5 patches; Fig. 2, E and F). Login to comment 127 ABCC7 p.Val317Gln i for V317Q CFTR was 0.59 Ϯ 0.01 pA at ϩ100 mV and 0.73 Ϯ 0.01 pA at -100 mV (P Ͻ 0.001). Login to comment 130 ABCC7 p.Val317Glu The rectification of V317E unitary currents was slight and in the opposite direction of the rectification observed in whole cell experiments. Login to comment 131 ABCC7 p.Val317Glu Thus rectification of V317E whole cell currents cannot be explained by voltage-dependent alterations in single-channel conductance. Login to comment 133 ABCC7 p.Val317Glu To compare the time-averaged, steady-state currents in patches containing either WT or V317E CFTR at multiple holding potentials, it was necessary to control for differences in channel number (N) between patches. Login to comment 146 ABCC7 p.Val317Glu ABCC7 p.Val317Gln Unitary current (i)-V relationships for WT, V317E, and V317Q CFTR. Login to comment 150 ABCC7 p.Val317Glu C: closed-to-open transition of V317E CFTR at ϩ80 (top) and -80 (bottom) mV. Login to comment 152 ABCC7 p.Val317Glu D: i-V relationship for V317E CFTR was fitted with 2 linear functions yielding g ϭ 7.65 Ϯ 0.14 pS at Vm ϭ -100 to 0 mV and g ϭ 6.34 Ϯ 0.14 pS at Vm ϭ 0 to ϩ100 mV (n ϭ 7 patches). Login to comment 153 ABCC7 p.Val317Gln ABCC7 p.Val317Gln E: closed-to-open transition of V317Q CFTR at ϩ80 (top) and -80 (bottom) mV. Login to comment 155 ABCC7 p.Val317Gln F: i-V relationship for V317Q CFTR was fitted with 2 linear functions yielding g ϭ 7.28 Ϯ 0.16 pS at Vm ϭ -100 to 0 mV and g ϭ 5.91 Ϯ 0.06 pS at Vm ϭ 0 to ϩ100 mV (n ϭ 5 patches). Login to comment 167 ABCC7 p.Val317Glu However, this was not the case for V317E CFTR. Login to comment 171 ABCC7 p.Val317Gln The behavior of V317Q CFTR channels was also examined at multiple potentials. Login to comment 174 ABCC7 p.Val317Glu Mechanism of the voltage dependence of V317E CFTR. Login to comment 175 ABCC7 p.Val317Glu Comparison of V317E and WT CFTR revealed markedly different single-channel kinetics (Fig. 5A). Login to comment 182 ABCC7 p.Val317Glu For V317E CFTR, burst durations were significantly reduced compared with WT CFTR over the entire voltage range (Fig. 5B). Login to comment 183 ABCC7 p.Val317Glu ABCC7 p.Val317Glu However, the difference between V317E and WT CFTR burst durations cannot explain the voltage dependence of Po observed in the mutant, because the burst durations of V317E CFTR at negative potentials were not significantly shorter than the burst durations at positive potentials. Login to comment 184 ABCC7 p.Val317Glu Closed times for either WT or V317E CFTR were fairly consistent in any single experiment, but they varied from patch to patch, probably as a result of variable phosphorylation levels. Login to comment 197 ABCC7 p.Val317Glu ABCC7 p.Val317Glu L139VOLTAGE-SENSITIVE GATING OF V317E CFTR AJP-Lung Cell Mol Physiol • VOL 282 • JANUARY 2002 • www.ajplung.org ing V317E CFTR revealed significant voltage dependence in the closed duration, with closed times being 4.50 Ϯ 0.93-fold longer (P Ͻ 0.05) at Vm ϭ -100 mV compared with those at Vm ϭ ϩ100 mV in the same patch (n ϭ 4). Login to comment 202 ABCC7 p.Val317Glu To test this possibility, we observed the voltage dependence of V317E CFTR activity at 1 and 10 mM ATP. Login to comment 205 ABCC7 p.Val317Glu Thus the effect of mutation V317E on channel gating does not appear to change the ATP dependence at concentrations in excess of the reported dissociation constant (29). Login to comment 207 ABCC7 p.Val317Glu To determine whether the voltage dependence of Po in V317E CFTR was influenced by an anion gradient, we repeated our experiments using a low-Cl- (15 mM) solution in the pipette. Login to comment 211 ABCC7 p.Val317Glu Comparing V317E CFTR channel behavior at ϩ100 and -100 mV (Fig. 7D, top and bottom, respectively), one can see a reduced i at ϩ100 mV, much like that seen in WT CFTR. Login to comment 213 ABCC7 p.Val317Glu The i-V relationship for V317E CFTR was shifted to the right by 49 mV (58.8 mV after corrections) in the presence of our low-Cl- pipette solution (Fig. 7E). Login to comment 214 ABCC7 p.Val317Glu ABCC7 p.Val317Glu The i-V of V317E CFTR in these conditions showed modestly increased inward rectification compared with that of the WT channel due to the reduced single-channel amplitude at positive potentials (Fig. 2D), V317E CFTR i amplitude at the most depolarized voltage being only 85% of that seen in WT CFTR. Login to comment 217 ABCC7 p.Val317Glu ABCC7 p.Val317Gln Time-averaged I-V relationships for WT, V317E, and V317Q CFTR. Login to comment 219 ABCC7 p.Val317Glu B: typical WT CFTR currents at positive (ϩ100 mV; top) and negative (-100 mV; bottom) potentials. C: mean I-V relationship for V317E CFTR shows a marked outward rectification. Login to comment 221 ABCC7 p.Val317Glu D: typical V317E CFTR currents at positive (ϩ100 mV; top) and negative (-100 mV; bottom) potentials. Login to comment 222 ABCC7 p.Val317Gln E: mean I-V relationship for V317Q CFTR is linear. Login to comment 223 ABCC7 p.Val317Gln F: typical V317Q CFTR currents at positive and negative potentials. Login to comment 226 ABCC7 p.Val317Glu L140 The time-averaged I-V relationship for V317E CFTR in the asymmetrical Cl- solutions showed a pronounced outward rectification (Fig. 7F), whereas that for WT CFTR remained nearly linear (Fig. 7C). Login to comment 228 ABCC7 p.Val317Glu Therefore, similar to the V317E CFTR behavior characterized in symmetrical Cl- , we conclude that outward rectification of I in the presence of a Cl-gradient is likely due to a voltage dependence of Po. Login to comment 231 ABCC7 p.Val317Glu In symmetrical Cl- there is an obvious voltage dependence in NPo for V317E CFTR (Fig. 8A) that was most pronounced at Vm values more positive than 0 mV, where net Cl- movement is inward. Login to comment 237 ABCC7 p.Val317Glu This insensitivity (to the direction of ion movement) of the voltage dependence of NPo for V317E CFTR argues strongly against voltage-dependent blockade of the pore. Login to comment 240 ABCC7 p.Val317Glu Single-channel kinetics of V317E vs. WT CFTR. Login to comment 241 ABCC7 p.Val317Glu A: single-channel records of V317E and WT CFTR at Vm ϭ -100 and ϩ100 mV. Login to comment 242 ABCC7 p.Val317Glu ABCC7 p.Val317Glu B: mean burst duration of V317E (E) and WT (F) CFTR over a range of potentials. C: mean closed durations in paired experiments at ϩ100 and -100 mV for WT (F) and V317E (E) CFTR. Login to comment 246 ABCC7 p.Val317Glu Values are means Ϯ SD; n ϭ 40-454 bursts at each potential for WT CFTR and n ϭ 17-47 bursts for V317E CFTR. Login to comment 247 ABCC7 p.Val317Glu ABCC7 p.Val317Glu L141VOLTAGE-SENSITIVE GATING OF V317E CFTR AJP-Lung Cell Mol Physiol • VOL 282 • JANUARY 2002 • www.ajplung.org V317E CFTR currents was due to voltage-dependent kinetics of opening and closing. Login to comment 250 ABCC7 p.Val317Glu To determine whether the process evident in V317E CFTR was similar to voltage-dependent gating mechanisms observed in other channels, we asked whether the kinetics of the relaxations at hyperpolarizing potentials were sensitive to Vm. Login to comment 252 ABCC7 p.Val317Glu Relaxations of inward currents were fitted with a second-order exponential in V317E CFTR, whereas only a first-order exponential was required for WT CFTR currents. Login to comment 253 ABCC7 p.Val317Glu Time constants for these relaxations in V317E CFTR were strongly dependent on voltage (values for the longer component, ␶2, were 157.0 Ϯ 10.7 ms at -120 mV and 294.2 Ϯ 18.2 ms at -60 mV; n ϭ 5 patches; P Ͻ 0.001 by paired t-test). Login to comment 254 ABCC7 p.Val317Gln The kinetic behavior of V317Q-CFTR Fig. 6. Login to comment 255 ABCC7 p.Val317Glu Increasing ATP concentration ([ATP]) 10-fold did not overcome the voltage-dependent decrease in opening rate in V317E CFTR. Login to comment 256 ABCC7 p.Val317Glu A: V317E CFTR activity in the presence of either 1 or 10 mM ATP in the same patch. Login to comment 258 ABCC7 p.Val317Glu B: multiple of increase in time-averaged V317E CFTR current when jumping from -100 to ϩ100 mV in the presence of 1 or 10 mM ATP. Login to comment 260 ABCC7 p.Val317Glu C: Vm protocol, which was applied in the presence of each [ATP] in A. Fig. 7. i-V and I-V relationships for WT and V317E CFTR in the presence of asymmetrical Cl- . Login to comment 264 ABCC7 p.Val317Glu D: typical V317E CFTR currents at positive (ϩ100 mV) and negative (-100 mV) potentials in asymmetrical Cl- . Login to comment 265 ABCC7 p.Val317Glu E: mean i-V relationship for V317E CFTR. Login to comment 266 ABCC7 p.Val317Glu F: mean I-V relationship for V317E CFTR. Login to comment 270 ABCC7 p.Val317Glu L142 VOLTAGE-SENSITIVE GATING OF V317E CFTR AJP-Lung Cell Mol Physiol • VOL 282 • JANUARY 2002 • www.ajplung.org onAugust,2011ajplung.physiology.orgDownloadedfrom macroscopic currents did not differ from WT CFTR (data not shown). Login to comment 275 ABCC7 p.Val317Glu Oocytes expressing V317E CFTR exhibited an increased conductance at ϩ50 mV as the prepulse duration was lengthened (Fig. 9A). Login to comment 278 ABCC7 p.Val317Glu The slower component of the relaxation (␶2) in V317E CFTR currents contributed more to the fit as the prepulse duration was lengthened from 20 to 250 ms. Login to comment 285 ABCC7 p.Val317Glu The voltage-dependent gating of V317E CFTR was evident both in relaxations of macroscopic currents and in the altered gating of single channels. Login to comment 290 ABCC7 p.Val317Glu ABCC7 p.Val317Gln Relative NPo was fairly constant over the tested voltage range (-100 to ϩ100 mV) for WT and V317Q CFTR, whereas relative NPo for V317E CFTR displayed a marked voltage dependence. Login to comment 293 ABCC7 p.Val317Glu V317E CFTR exhibited a voltage-dependent increase in NPo at positive potentials, even when the direction of net Cl- movement was reversed (i.e., between Vm ϭ 0 and ϩ40 mV). Login to comment 296 ABCC7 p.Val317Glu Macroscopic currents in V317E CFTR exhibit voltage-jump relaxations. Login to comment 297 ABCC7 p.Val317Glu A: whole cell currents in 1 oocyte expressing V317E CFTR. Login to comment 300 ABCC7 p.Val317Glu Relaxations at -120 mV in V317E CFTR were fitted best with a second-order exponential (Simplex fitting algorithm; SD was typically approximately Յ0.01). Login to comment 303 ABCC7 p.Val317Glu D: dependence of the time constants on duration of the prepulse to ϩ50 mV (n ϭ 8 and 5 cells for V317E and WT CFTR, respectively). Login to comment 306 ABCC7 p.Val317Glu Slower component (␶2) in V317E CFTR showed a clear dependence on prepulse duration (␶2 ϭ 131.5 Ϯ 8.6 vs. 205.1 Ϯ 31.6 ms for 50- and 200-ms prepulses, respectively; P Ͻ 0.001). Login to comment 309 ABCC7 p.Val317Glu L143VOLTAGE-SENSITIVE GATING OF V317E CFTR AJP-Lung Cell Mol Physiol • VOL 282 • JANUARY 2002 • www.ajplung.org the strong outward rectification of macroscopic currents carried by this channel. Login to comment 314 ABCC7 p.Gly314Glu ABCC7 p.Lys335Glu ABCC7 p.Thr1134Glu ABCC7 p.Ser341Glu ABCC7 p.Gly91Glu Other glutamate substitutions in TM domains 1, 5, 6, and 12 have been investigated [G91E, G314E, and K335E (16); S341E and T1134E (20)], but none of these exhibited voltage-dependent gating (McCarty and Dawson, unpublished observations). Login to comment 317 ABCC7 p.Val317Gln Introduction of a similarly sized but uncharged amino acid side chain at this position (V317Q CFTR) did not affect gating, indicating that it is the charge, not the bulkiness of the side chain, that confers the voltage-dependent behavior. Login to comment 319 ABCC7 p.Val317Glu ABCC7 p.Val317Gln The small change in single-channel conductance was found for both V317E and V317Q-CFTR, indicating that it does not result from electrostatic repulsion due to introduction of the negative charge. Login to comment 322 ABCC7 p.Val317Glu The voltage dependence induced by the V317E mutation impacts the CFTR gating scheme at a point distal to the binding and hydrolysis of ATP at the NBDs, as evidenced by the insensitivity of the opening rate to increased [ATP]. Login to comment 331 ABCC7 p.Val317Glu The ability to confer voltage dependence to the gating mechanism, as seen with the V317E mutation, may provide a means to localize the structures that comprise the gate. Login to comment