ABCMdb

PMID: 12411425

Gong X, Burbridge SM, Lewis AC, Wong PY, Linsdell P
Mechanism of lonidamine inhibition of the CFTR chloride channel.
Br J Pharmacol. 2002 Nov;137(6):928-36., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Arg334Cys 5 Several point mutations within the sixth transmembrane region of CFTR (R334C, F337S, T338A and S341A) signi®cantly weakened block of macroscopic CFTR current, suggesting that lonidamine enters deeply into the channel pore from its intracellular end. Login to comment 116 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys As shown in Figure 7a, 55 mM lonidamine inhibited currents carried by R334C, K335A, F337S, T338A and S341A-CFTR. Login to comment 117 ABCC7 p.Ser341Ala ABCC7 p.Phe337Ser ABCC7 p.Arg334Cys However, R334C, F337S and S341A were only weakly inhibited by this concentration relative to wild-type CFTR (see Figure 1). Login to comment 118 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys The eect of these mutations on block by lonidamine is more clearly seen in the dose-response curves shown in Figure 7b. Fits of these mean data by equation 1 suggests a Kd (at 7100 mV) of 58.5 mM for wild-type, 65.6 mM for K335A, 90.0 mM for T338A, 186 mM for F337S, 206 mM for S341A, and 338 mM for R334C. Login to comment 119 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Arg334Cys Similar analyses at other potentials showed a similar increase in Kd in R334C, F337S, S341A and (to a far lesser extent) T338A (Figure 7c). Login to comment 120 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys Fitting data from individual patches with equation 2 gave similar and, except in the case of K335A, signi®cant changes in Kd(-100): wild-type 60.6+5.2 mM (n=5), K335A 63.1+7.4 mM (n=5) (P40.05), T338A 93.4+4.1 mM (n=5) (P50.002), F337S 166+18 mM (n=5) (P50.0005), S341A 169+25 mM (n=5) (P50.005), R334C 260+19 mM (n=4) (P50.00001). Login to comment 121 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys ABCC7 p.Arg334Cys These same ®ts also revealed changes in the voltage dependence of block, as judged by changes in d, although this was only statistically signi®cant in the case of R334C: wild-type 0.426+0.033 (n=5), K335A 0.484+0.024 (n=5) (P40.05), T338A 0.410+0.045 (n=5) (P40.05), F337S 0.365+0.015 (n=5) (P40.05), S341A 0.285+0.061 (n=5) (P40.05), R334C 0.233+0.066 (n=4) (P50.05). Login to comment 143 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys (a) Example I-V relationships for R334C, K335A, F337S, T338A and S341A-CFTR, before (solid lines) and following (dotted lines) addition of 55 mM lonidamine to the intracellular solution. Login to comment 145 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys (b) Concentration dependence of block at 7100 mV for wild-type, R334C, K335A, F337S, T338A and S341A. Login to comment 147 ABCC7 p.Ser341Ala ABCC7 p.Thr338Ala ABCC7 p.Phe337Ser ABCC7 p.Lys335Ala ABCC7 p.Arg334Cys Each has been ®tted by equation 1, giving Kds of 58.5 mM (wild-type), 65.6 mM (K335A), 90.0 mM (T338A), 186 mM (F337S), 206 mM (S341A) and 338 mM (R334C). Login to comment 154 ABCC7 p.Ser341Ala ABCC7 p.Phe337Ser ABCC7 p.Arg334Cys Lonidamine block was weakened in the TM6 mutants R334C, F337S and S341A (Figure 7), suggesting that these residues may normally contribute to lonidamine binding within the pore. Login to comment 157 ABCC7 p.Arg334Cys The strong eects of the R334C mutant (Figure 7) are somewhat surprising, given that this residue is purportedly in the external pore vestibule. Login to comment