ABCMdb

PMID: 1281220

Sheppard DN, Welsh MJ
Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.
J Gen Physiol. 1992 Oct;100(4):573-91., [PubMed]

Sentences

No. Mutations Sentence Comment

46 ABCC7 p.Lys1250Met MATERIALS AND METHODS For this study we used NIH 3T3 fibroblasts that had been stably infected with a retrovirus expressing either wild-type human CFTR (Gregory, Cheng, Rich, Marshall, Paul, Hehir, Ostedgaard, Klinger, Welsh, and Smith, 1990) or a CFTR mutant in NBD2 (CFIR-KI250M, in which lysine 1250 was changed to methionine) (Anderson et al., 1991a). Login to comment 48 ABCC7 p.Lys335Glu We also transiently expressed another mutant (CFI'R-K335E, in which lysine 335 in the first transmembrane domain was changed to glutamic acid) in HeLa cells using the vaccinia virus-T7 hybrid expression system (Rich et al., 1990; Elroy-Stein, Fuerst, and Moss, 1989). Login to comment 161 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met We therefore examined the effect of glibenclamide, the most potent inhibitor we had identified, on CI- currents generated by several CFFR mutants. We studied 100 • 1992 CFTR containing mutations that affect each of the three types of domains of CFTR: CFFP~R where part of the R domain has been deleted (amino acids 708-835); CVFR-K335E, which contains a mutation in the sixth putative membrane-spanning sequence; and CFTR-K1250M, which contains a mutation in the second NBD. Login to comment 171 ABCC7 p.Lys335Glu CFFR-K335E forms CI- channels that are similar to wild-type channels, except that the anion selectivity is altered such that I- > Br- > C1- and currents show some outward rectification (Anderson et al., 1991b and Fig. 7, D and F). Login to comment 172 ABCC7 p.Lys335Glu Glibenclamide inhibited CFTR-K335E CI- currents, and the effect was similar to that observed with wild-type CFTR (Fig. 7, E and F). Login to comment 173 ABCC7 p.Lys1250Met Comparison with ATP-dependent transporters suggest that mutations in the second NBD, such as CFTR-K1250M, are likely to abolish or impair the function of SHEPP~LRDANDWELSHK-ATP Channel Regulators Inhibit CFTR NBD2 (Anderson et al., 1991a). Login to comment 174 ABCC7 p.Lys1250Met Nevertheless, CFFR-K1250M produces CI- channels that have cAMP-dependent regulation and biophysical properties that are similar to wild-type CFFR (see Anderson et al., 1991a, and Fig. 7, G and I). Login to comment 175 ABCC7 p.Lys1250Met Inhibition of CFTR-K1250M CI- currents by glibenclamide was similar to that observed with wild-type CYI'R (Fig. 7, H and I). Login to comment 176 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met +SO mV 0 m V ~ -go mV CFTFI~ A~ ......~-1 baseline D CFTR-K335E I ii..i.Iii cAMP .... 13, CFTR-K1250M 1cAMP baseline + gllbenclamlde 250 pA L 50 ms C 1500 1000 5OO -500 o1~0 -100 -75 -50 -25 0 V (mV) ---o-- baseline ---4-- gllbenclamlde E) I cAMP + gllbenclamlde / tp----e F 1000 750 5OO -100 -75 -50 -25 0 V (mV) H ) 1000 PAL__ 50 ms I i cAMP + gllbenclamlde --o-- cAMP --e-- gllbanclamlde / _~_ _ 4OOO 3OOO ~q]O0 I000 0 I000 2OOO 30OO mOO -100 -75 -50 -25 0 V (mV) 25 50 25 50 25 50 FIGURE 7. Login to comment 178 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met Traces are from C127 cells stably expressing CFTP-~R (A-C), HeLa cells transiently expressing CFFR-K335E (D-F), and NIH 3T3 cells stably expressing CFIR-K1250M (G-I). Login to comment 180 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met CFFRAR currents were recorded in the absence of cAMP, but for CFFR-K335E and CFTR-K1250M currents cAMP agonists were present. Login to comment 185 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met Data for steady-state current values measured at +50 and -90 mV are shown for wild-type CF-FR (A), CFTRAR (B), CFFR-K335E (C), and CFTR-K1250M (D). Login to comment 186 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met The dose-response curves for wild-type CFFR, CFTR-K335E, and CFTR-K1250M were similar. Login to comment 187 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met Although the effect was not marked, in each case inhibition was slightly more potent at -90 mV than at +50 inV. A 100 8O O 60 c g 20 B 100 8O o 6o eo o m 20 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992 C CFTR 100 ' --o-- 50mV 80 e 6o C g,m 2O 0 20 40 60 80 1C10 0 gllbenclamlde (,aM) D CFTRAR 100 80 2 60 c i 2O 1 ~ ~ 866o o glil~nclamlde (gM) CFTR-K335E 0 20 40 ~ 80 100 glibenclamlda (,ttM) CFTR-K1250M "il o 2i~ ,o 6i) 8o lOO glibenclamlde (i.tM) FIGURE 8. Login to comment 191 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met (A) Wild-type CFTR; (B) CFTR~R; (C) CFTR-K335E; (D) CFFR-K1250M. Login to comment 196 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met Half-maximal inhibition by glibenclamide was similar in all cases, occurring at ~ 20 TABLE I1 Effect of Glibenclamide on CFTR Mutants 4-50 mV -90 mV Mutant Experiments Ki n Ki n my/ u2v/ CFTR 21.8 ± 5.2 0.8 - 0.1 19.0 - 5.6* 0.7 ± 0.1 7 CFTR-K335E 25.9 ± 4.1 0.9 ± 0.I 20.4 +--3.7* 0.9 ± 0.1 6 CFTR-K1250M 16.8 _ 4.2 0.8 ± 0.1 31.2 ± 2.5 1.0 - 0.1 5 Ki and n values were calculated as described in Table I for currents measured at +50 and -90 mV for the number of experiments listed. Login to comment 199 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met Half-maximal inhibition by glibenclamide, measured at -90 mV, showed a small but statistically significant increase in potency compared with that at +50 mV for wild-type CFTR and CFTR-K335E, but not CFFR-K1250M. Login to comment 248 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met Inhibition of CFTR-K335E and CFTR-K1250M CI- currents by glibenclamide resembled that of wild-type CFFR. Login to comment 249 ABCC7 p.Lys335Glu ABCC7 p.Lys1250Met This suggests that residues K335E and K1250M do not form a critical part of the glibenclamide interaction site. Login to comment