ABCMdb

ABCC7 p.Val317Glu

None has been submitted yet.

Publications

PMID: 11741825 [PubMed] Zhang ZR et al: "Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR."

No. Sentence Comment

8 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 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 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 V317E CFTR macroscopic currents display marked rectification. Login to comment
23 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 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 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 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 V317E CFTR also exhibited modest tail currents (Fig. 1B) not seen in WT CFTR with this voltage protocol. Login to comment
99 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 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 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 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 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 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 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 B: family of currents for an oocyte expressing V317E CFTR. Login to comment
117 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 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 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 Thus i at positive potentials for V317E CFTR exhibited only 87% of the conductance of WT CFTR. Login to comment
130 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 Thus rectification of V317E whole cell currents cannot be explained by voltage-dependent alterations in single-channel conductance. Login to comment
133 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 Unitary current (i)-V relationships for WT, V317E, and V317Q CFTR. Login to comment
150 C: closed-to-open transition of V317E CFTR at ϩ80 (top) and -80 (bottom) mV. Login to comment
152 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
167 However, this was not the case for V317E CFTR. Login to comment
174 Mechanism of the voltage dependence of V317E CFTR. Login to comment
175 Comparison of V317E and WT CFTR revealed markedly different single-channel kinetics (Fig. 5A). Login to comment
182 For V317E CFTR, burst durations were significantly reduced compared with WT CFTR over the entire voltage range (Fig. 5B). Login to comment
183 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 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 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 To test this possibility, we observed the voltage dependence of V317E CFTR activity at 1 and 10 mM ATP. Login to comment
205 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 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 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 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 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 Time-averaged I-V relationships for WT, V317E, and V317Q CFTR. Login to comment
219 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 D: typical V317E CFTR currents at positive (ϩ100 mV; top) and negative (-100 mV; bottom) potentials. Login to comment
226 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 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 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 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 Single-channel kinetics of V317E vs. WT CFTR. Login to comment
241 A: single-channel records of V317E and WT CFTR at Vm ϭ -100 and ϩ100 mV. Login to comment
242 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 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 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 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 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 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
255 Increasing ATP concentration ([ATP]) 10-fold did not overcome the voltage-dependent decrease in opening rate in V317E CFTR. Login to comment
256 A: V317E CFTR activity in the presence of either 1 or 10 mM ATP in the same patch. Login to comment
258 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 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 D: typical V317E CFTR currents at positive (ϩ100 mV) and negative (-100 mV) potentials in asymmetrical Cl- . Login to comment
265 E: mean i-V relationship for V317E CFTR. Login to comment
266 F: mean I-V relationship for V317E CFTR. Login to comment
270 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 Oocytes expressing V317E CFTR exhibited an increased conductance at ϩ50 mV as the prepulse duration was lengthened (Fig. 9A). Login to comment
278 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 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 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 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 Macroscopic currents in V317E CFTR exhibit voltage-jump relaxations. Login to comment
297 A: whole cell currents in 1 oocyte expressing V317E CFTR. Login to comment
300 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 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 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 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
319 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 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 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

PMID: 15711774 [PubMed] Zhang ZR et al: "Time-dependent interactions of glibenclamide with CFTR: kinetically complex block of macroscopic currents."

No. Sentence Comment

300 Unblocked macroscopic currents in WT-CFTR and nearly all mutants studied to date are time-independent (exceptions are S1118F-CFTR and V317E-CFTR, which confer voltage-jump relaxations on currents in the absence of added blockers; Zhang, McDonough & McCarty, 2000b; Zhang et al. 2002). Login to comment