ABCMdb

PMID: 9463368

Sugita M, Yue Y, Foskett JK
CFTR Cl- channel and CFTR-associated ATP channel: distinct pores regulated by common gates.
EMBO J. 1998 Feb 16;17(4):898-908., [PubMed]

Sentences

No. Mutations Sentence Comment

115 ABCC7 p.Arg347Glu We confirmed the reduced single channel conductance of R347E CFTR (1.6 Ϯ 0.1 pS; nϭ3, p Ͻ0.01) (Figure 7C). Login to comment 117 ABCC7 p.Arg347Glu Effects of DIDS and R347E mutation on CFTR Cl- channels and CFTR-associated ATP channels. Login to comment 121 ABCC7 p.Arg347Glu ABCC7 p.Arg347Glu (C) Current traces from a MDCK cell expressing R347E in an inside-out patch with 100 mM ATP in the pipette and 140 mM Clin the bath. Login to comment 122 ABCC7 p.Arg347Glu R347E mutation had little effect on the slope conductance of the CFTR-associated ATP channels (5.08 Ϯ 0.27 pS; nϭ3) (Figure 7C). Login to comment 123 ABCC7 p.Arg347Glu Effects of DIDS and R347E mutation on CFTR Cl-channels and CFTR-associated ATP channels. Login to comment 128 ABCC7 p.Arg347Glu ABCC7 p.Ser660Ala We first examined the deletion mutant CFTR"06;R-S660A, Fig. 8. Login to comment 129 ABCC7 p.Arg347Glu R347E mutation had little effect on the slope conductance of the CFTR-associated ATP channels (5.08 afe; 0.27 pS; nafd;3) (Figure 7C). Login to comment 131 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Ser660Ala The residues altered in CFTR∆R-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment 132 ABCC7 p.Ser660Ala (B) Current traces from a MDCK cell expressing CFTR∆R-S660A in the presence or absence of PKA at various membrane potentials (representative of five independently observed channels). Login to comment 135 ABCC7 p.Ser660Ala We first examined the deletion mutant CFTRƊR-S660A, Fig. 8. Login to comment 137 ABCC7 p.Ser660Ala which lacks much of the R domain and replaces Ser-660 with alanine (Figure 8A). Login to comment 138 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Ser660Ala The residues altered in CFTRƊR-S660A, CFTR S-oct-D, K464A and K1250A mutants are shown. Login to comment 139 ABCC7 p.Ser660Ala ABCC7 p.Ser660Ala The CFTR∆R-S660A Cl- channels were active in MDCK cells (Po ϭ 0.05 Ϯ 0.003, nϭ3) requiring the presence of 903 cytosolic ATP alone (Figure 8B), and the gating activity did not increase following addition of PKA, in agreement with the previous results. Login to comment 140 ABCC7 p.Ser660Ala However, the CFTR∆R-S660A mutant eliminated CFTR-associated ATP channel activities, even in the presence of both PKA and ATP (nϭ5; Figure 8B). Login to comment 142 ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp To examine this further, we expressed CFTR S-oct-D, which contains eight serine-to-aspartate substitutions in the R domain (S660D, S686D, S700D, S712D, S737D, S768D, S795D and S813D) (Figure 8A). Login to comment 145 ABCC7 p.Ser660Ala which lacks much of the R domain and replaces Ser-660 with alanine (Figure 8A). Login to comment 147 ABCC7 p.Ser660Ala The CFTRƊR-S660A Cl-channels were active in MDCK cells (Po afd; 0.05 afe; 0.003, nafd;3) requiring the presence of 903 cytosolic ATP alone (Figure 8B), and the gating activity did not increase following addition of PKA, in agreement with the previous results. Login to comment 148 ABCC7 p.Ser660Ala However, the CFTRƊR-S660A mutant eliminated CFTR-associated ATP channel activities, even in the presence of both PKA and ATP (nafd;5; Figure 8B). Login to comment 150 ABCC7 p.Ser737Asp ABCC7 p.Ser768Asp ABCC7 p.Ser686Asp ABCC7 p.Ser795Asp ABCC7 p.Ser660Asp ABCC7 p.Ser712Asp ABCC7 p.Ser700Asp ABCC7 p.Ser813Asp To examine this further, we expressed CFTR S-oct-D, which contains eight serine-to-aspartate substitutions in the R domain (S660D, S686D, S700D, S712D, S737D, S768D, S795D and S813D) (Figure 8A). Login to comment 155 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala These NBD1 and NBD2 mutants, containing the individual mutations K464A and K1250A, respectively, were expressed in MDCK cells and single-channel currents of CFTR Cl- channels and CFTR-associated ATP channels were analyzed. Login to comment 161 ABCC7 p.Lys464Ala the K464A mutant exhibited no significant differences in Po (p Ͼ0.05), to (p Ͼ0.05) and the mean closed time (tc) (p Ͼ0.15), although there was a tendency towards increased tc and shorter to (Figure 10B, C, D and E). Login to comment 163 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Lys464Ala The K464A mutation similarly had no significant effects on the gating of the CFTR-associated ATP channels (Figure 10B, C, D and E). Login to comment 164 ABCC7 p.Lys1250Ala In contrast, mutation of the corresponding lysine in NBD2 (K1250A) resulted in CFTR-associated ATP channels (p Ͻ0.01) as well as CFTR Cl- channels (p Ͻ0.02) that exhibited significantly prolonged to (Figure 10A and D). Login to comment 165 ABCC7 p.Lys1250Ala The K1250A mutation resulted in gating behaviors of wild-type CFTR Cl- channels and CFTR-associated ATP channels which mimicked those induced by non-hydrolyzable nucleotide analogues (Figure 10A). Login to comment 166 ABCC7 p.Lys1250Ala In addition, we noted that the K1250A mutation also decreased tc of both CFTR Cl- channels (p Ͻ0.01) as well as CFTR-associated ATP channels (p Ͻ0.01) (Figure 10E). Login to comment 167 ABCC7 p.Lys1250Ala As a result, the K1250A mutation caused a substantial increase in the Po of both channels (p Ͻ0.01) (Figure 10C). Login to comment 170 ABCC7 p.Lys464Ala the K464A mutant exhibited no significant differences in Po (p b0e;0.05), to (p b0e;0.05) and the mean closed time (tc) (p b0e;0.15), although there was a tendency towards increased tc and shorter to (Figure 10B, C, D and E). Login to comment 171 ABCC7 p.Lys1250Ala (A) Current traces from a MDCK cell expressing K1250A in an inside-out patch with 100 mM ATP in the pipette and 140 mM Clin the bath at various membrane potentials (representative of six independently observed channels). Login to comment 172 ABCC7 p.Lys464Ala The K464A mutation similarly had no significant effects on the gating of the CFTR-associated ATP channels (Figure 10B, C, D and E). Login to comment 173 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala (B) Current traces from a MDCK cell expressing K464A at various membrane potentials (representative of nine independently observed channels). Login to comment 174 ABCC7 p.Lys1250Ala The K1250A mutation resulted in gating behaviors of wild-type CFTR Cl-channels and CFTR-associated ATP channels which mimicked those induced by non-hydrolyzable nucleotide analogues (Figure 10A). Login to comment 175 ABCC7 p.Lys1250Ala In addition, we noted that the K1250A mutation also decreased tc of both CFTR Cl-channels (p b0d;0.01) as well as CFTR-associated ATP channels (p b0d;0.01) (Figure 10E). Login to comment 176 ABCC7 p.Lys1250Ala As a result, the K1250A mutation caused a substantial increase in the Po of both channels (p b0d;0.01) (Figure 10C). Login to comment 180 ABCC7 p.Lys1250Ala (A) Current traces from a MDCK cell expressing K1250A in an inside-out patch with 100 mM ATP in the pipette and 140 mM Clin the bath at various membrane potentials (representative of six independently observed channels). Login to comment 183 ABCC7 p.Lys464Ala (B) Current traces from a MDCK cell expressing K464A at various membrane potentials (representative of nine independently observed channels). Login to comment 189 ABCC7 p.Arg347Glu Third, mutation of a residue in the CFTR Cl- channel pore, R347E, had little effect on the conductance of CFTR-associated ATP channels, in contrast to its effect on the CFTR Cl- conductance. Login to comment 199 ABCC7 p.Arg347Glu Third, mutation of a residue in the CFTR Cl-channel pore, R347E, had little effect on the conductance of CFTR-associated ATP channels, in contrast to its effect on the CFTR Cl-conductance. Login to comment 205 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Mutations in the conserved Walker A motif lysines of NBD1 (K464A) and NBD2 (K1250A) are thought to attenuate ATP hydrolysis with minimal effect on ATP binding (Sung et al., 1988; Schneider et al., 1994; Carson et al., 1995). Login to comment 206 ABCC7 p.Lys464Ala Previous analyses of the K464A mutant indicated that it had a reduced Po due to an increased closed time, although these effects were not pronounced (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment 209 ABCC7 p.Lys1250Ala The K1250A mutation had more pronounced effects. Login to comment 211 ABCC7 p.Lys1250Ala The prolonged channel open time in the K1250A mutant suggests that ATP hydrolysis at NBD2 closes the channel, consistent with previous observations (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment 215 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala Mutations in the conserved Walker A motif lysines of NBD1 (K464A) and NBD2 (K1250A) are thought to attenuate ATP hydrolysis with minimal effect on ATP binding (Sung et al., 1988; Schneider et al., 1994; Carson et al., 1995). Login to comment 216 ABCC7 p.Lys464Ala Previous analyses of the K464A mutant indicated that it had a reduced Po due to an increased closed time, although these effects were not pronounced (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment 219 ABCC7 p.Lys1250Ala The K1250A mutation had more pronounced effects. Login to comment 221 ABCC7 p.Lys1250Ala The prolonged channel open time in the K1250A mutant suggests that ATP hydrolysis at NBD2 closes the channel, consistent with previous observations (Carson et al., 1995; Gunderson and Kopito, 1995). Login to comment 272 ABCC7 p.Arg347Glu ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Ser660Ala Acknowledgements We thank M.Welsh for providing the CFTR∆R-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment 282 ABCC7 p.Arg347Glu ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala ABCC7 p.Ser660Ala Acknowledgements We thank M.Welsh for providing the CFTRƊR-S660A and CFTR S-oct-D mutants, R.Kopito for providing the K1250A and K464A mutants, J.Engelhardt for providing the R347E mutant, U.Patel for her precious technical help and D.Mak for helpful discussions. Login to comment