ABCMdb

PMID: 16183882

Fuller MD, Zhang ZR, Cui G, McCarty NA
The block of CFTR by scorpion venom is state-dependent.
Biophys J. 2005 Dec;89(6):3960-75. Epub 2005 Sep 23., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCC7 p.Lys1250Ala The on-rate of venom binding for intraburst block could be modulated by changing CFTR activity with vanadate or adenylyl-imidodiphosphate, or by introducing the Walker A mutation K1250A. Login to comment 33 ABCC7 p.Lys1250Ala We also found that the potency of venom for intraburst inhibition was reduced in single-channel recordings of WT-CFTR channels with very high open probability or when the venom was applied to K1250A-CFTR channels. Login to comment 41 ABCC7 p.Lys1250Ala The mutant K1250A-CFTR construct was prepared with the QuikChange protocol (Stratagene, La Jolla, CA) using oligonucleotide-mediated mutagenesis. Login to comment 163 ABCC7 p.Lys1250Ala Mutations in NBD-B, such as K1250A, result in channels that follow the initial WT-CFTR gating steps; however, rates of ATP binding at NBD-B and hydrolysis of that ATP are greatly reduced (boxes). Login to comment 216 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala K1250A-CFTR channels exhibit a greatly reduced rate of ATP hydrolysis, resulting in channels that are open for tens of seconds; however, K1250A-CFTR channels also exhibit a reduced opening rate such that they remain in the C2 closed state longer than WT-CFTR channels (18). Login to comment 217 ABCC7 p.Lys1250Ala Studies were performed with K1250A-CFTR in multichannel patches with 50 U/mL PKA and 1 mM MgATP continuously present. Login to comment 227 ABCC7 p.Lys1250Ala However, results from studies with WT-CFTR in macropatch recordings (Fig. 1 C), as well as K1250A-CFTR in recordings of only a few channels (Fig. 4 B), suggested that the venom does not interact with the open state. Login to comment 239 ABCC7 p.Lys1250Ala (C) Representative single-channel recording of K1250A-CFTR before and during treatment with 0.1 mg/mL Lqh-pf venom. Login to comment 293 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala TABLE 1 Lqh-pf venom becomes a more effective blocker at lower CFTR channel activity Condition Venom dose (mg/mL) Control Po With venom Po Fractional inhibition n Wild-type (low Po)* 0.1 0.181 6 0.031 0.118 6 0.018y 31.4 6 6.96y 6 Wild-type (high Po)z 0.1 0.584 6 0.091 0.554 6 0.087 5.08 6 0.19 2 Wild-type (high Po)z 0.2 0.419 6 0.020 0.226 6 0.080 46.7 6 16.4 2 Wild-type 1 VO4 § 0.2 0.569 6 0.158§ 0.391 6 0.121y§ 31.9 6 6.32y§ 3 Wild-type 1 AMP-PNP§ 0.2 0.618 6 0.107§ 0.442 6 0.095y§ 29.5 6 5.22y§ 3 K1250A 0.1 0.772 6 0.079 0.751 6 0.074 2.66 6 0.59 3 Inhibition of CFTR by Lqh-pf venom was determined under several experimental conditions used to control channel-open probability. Login to comment 301 ABCC7 p.Lys1250Ala In addition, we also employed the Walker A mutant K1250A-CFTR, which gates open much like WT-CFTR, although the rate of ATP binding to NBD-B is somewhat reduced, whereas hydrolysis of ATP is greatly reduced, resulting in open bursts that are many tens of seconds in duration (Fig. 2, boxes). Login to comment 310 ABCC7 p.Lys1250Ala In control conditions, K1250A-CFTR channels remained almost entirely in the open state (Fig. 7 C, top), with few brief closures. Login to comment 312 ABCC7 p.Lys1250Ala Treatment of single K1250A-CFTR channels with 0.1 mg/mL Lqh-pf venom resulted in only 2.66 6 0.59% inhibition of channel activity (Po ¼ 0.772 6 0.079 vs. 0.751 6 0.074, n ¼ 3). Login to comment 313 ABCC7 p.Lys1250Ala Notably, the durations of the venom-induced intraburst blocked states in K1250A-CFTR and WT-CFTR with ATP 1 AMP-PNP, or WT-CFTR with ATP 1 vanadate, were similar to those observed in WT-CFTR with ATP alone (400-700 ms). Login to comment 315 ABCC7 p.Lys1250Ala The results described above suggested that Lqh-pf venom might be less effective at inhibiting K1250A-CFTR channels or WT-CFTR channels locked open by AMP-PNP or vanadate because those channels occupy the FC state less frequently. Login to comment 320 ABCC7 p.Lys1250Ala Similar results were seen when WT-CFTR channels were locked open with vanadate (Fig. 8 B) or when K1250A-CFTR channels were activated with MgATP (Fig. 8 C). Login to comment 323 ABCC7 p.Lys1250Ala All K1250A-CFTR openings were used. Login to comment 330 ABCC7 p.Lys1250Ala (C) Representative trace of K1250A-CFTR with 1 mM MgATP and 50 U/mL PKA continuously present before and during application of 0.1 mg/mL Lqh-pf venom. Login to comment 334 ABCC7 p.Lys1250Ala The largest increase in mean open time between intraburst closings was seen in K1250A-CFTR where the channels remained open for an average of 1580.9 6 106.8 ms (n ¼ 3 recordings, n ¼ 3 bursts, n ¼ 177 open duration events; p # 0.001 compared to WT-CFTR) between intraburst closings. Login to comment 340 ABCC7 p.Lys1250Ala Additionally, WT-CFTR channels that were locked in the open conformation by vanadate or AMP-PNP, and CFTR channels bearing the K1250A mutation, were inhibited by venom only at higher concentrations. Login to comment 356 ABCC7 p.Lys1250Ala (C) Representative trace of K1250A-CFTR from an excised inside-out patch with 1 mM MgATP and 50 U/mL PKA continuously present. Login to comment 357 ABCC7 p.Lys1250Ala ABCC7 p.Lys1250Ala All recordings at Vm ¼ ÿ100 mV. Note that brief closings, $5 ms in duration, occur less frequently when CFTR channel activity is manipulated by drug (vanadate or AMP-PNP) or mutation (K1250A), compared to WT-CFTR channels that are activated by MgATP and PKA only. Login to comment 358 ABCC7 p.Lys1250Ala (D) Effect of vanadate, AMP-PNP or K1250A mutation on the frequency of intraburst closings in records filtered at 500 Hz. Login to comment 379 ABCC7 p.Lys1250Ala Furthermore, K1250A-CFTR channels and WT-CFTR channels locked open by AMP-PNP basically never reach the C4 state, and yet do show the lengthening of interburst durations (Fig. 4). Login to comment 391 ABCC7 p.Lys1250Ala The effect oninterburst kinetics cannot fully explain the reduced efficacy of inhibition of single K1250A-CFTR channels or single WT-CFTR channels in the presence of AMP-PNP or vanadate. Login to comment 400 ABCC7 p.Lys1250Ala This was most apparent in experiments with channels locked open by vanadate, AMP-PNP, or mutation K1250A. Login to comment 424 ABCC7 p.Lys1250Ala The previous results could also explain why Lqh-pf venom is significantly less effective when applied to K1250A-CFTR, since the frequency of short intraburst closings in this mutant is much less than that seen with WT-CFTR (Fig. 8 D). Login to comment 439 ABCC7 p.Lys1250Ala The K1250A-CFTR results are also consistent with this notion. Login to comment 441 ABCC7 p.Lys1250Ala As a result, the NBD-ICD interaction would be stabilized, resulting in a decrease in the frequency of FC intraburst closings during open bursts in K1250A-CFTR, which is what we see in Fig. 8 C. The structural differences that are dependent on the nucleotide species bound at NBD-B could also have a large effect on the on-rate of venom binding during the FC intraburst closings. Login to comment 442 ABCC7 p.Lys1250Ala This would explain the decrease in intraburst inhibition of K1250A-CFTR channels and of WT-CFTR channels that are locked open with AMP-PNP or vanadate when a low concentration of the venom was applied (Fig. 7). Login to comment 446 ABCC7 p.Lys1250Ala K1250A-CFTR with ATP) (Table 1) is similar to the predicted order shown in Fig. 8 D. Small conformational changes in the NBD dimer are needed to signal for channel opening (9). Login to comment