ABCMdb

PMID: 11788611

Berger AL, Ikuma M, Hunt JF, Thomas PJ, Welsh MJ
Mutations that change the position of the putative gamma-phosphate linker in the nucleotide binding domains of CFTR alter channel gating.
J Biol Chem. 2002 Jan 18;277(3):2125-31., 2002-01-18 [PubMed]

Sentences

No. Mutations Sentence Comment

4 ABCC7 p.Gln493Ala In NBD1, Q493A reduced the frequency of channel opening, suggesting a role for this residue in coupling ATP binding to channel opening. Login to comment 5 ABCC7 p.Asn505Cys In contrast, N505C increased the frequency of channel opening, consistent with a role for Asn-505 in stabilizing the inactive state of the NBD. Login to comment 6 ABCC7 p.Gln1291Ala In NBD2, Q1291A decreased the effects of pyrophosphate without altering other functions. Login to comment 22 ABCC7 p.Lys1250Ala ABCC7 p.Lys464Ala However, the effects of mutations in the two NBDs are not symmetrical; the K1250A mutation dramatically prolongs the burst duration, whereas the K464A mutation reduces the frequency of channel opening but does not change burst duration. Login to comment 79 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala Single-channel gating of wild type CFTR, CFTR-Q493A, and CFTR-Q1291A. Login to comment 80 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala A, examples of current from excised inside-out membrane patches containing single CFTR channels in the presence of 1 mM ATP and 75 nM PKA. Membrane potential was clamped at -80 mV. B, data from multiple patches. Asterisk indicates p Ͻ 0.05; n ϭ 7 for WT, n ϭ 4 for CFTR-Q493A, and n ϭ 3 for CFTR-Q1291A. Login to comment 93 ABCC7 p.Gln493Ala In contrast, the Q493A variant exhibited a substantially reduced Po because of a prolonged interburst interval with little change in FIG. 3. Login to comment 94 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala Wild type CFTR, CFTR-Q493A, and CFTR-Q1291A had similar dose response curves for ATP-stimulated Cl- current. Login to comment 98 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala The apparent EC50 was 176 Ϯ 67 ␮M for wild type CFTR, 217 Ϯ 55 ␮M for CFTR-Q493A, and 159 Ϯ 70 ␮M for CFTR-Q1291A. Login to comment 101 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala Wild type CFTR, CFTR-Q493A, and CFTR-Q1291A showed similar cation requirements for Cl-channel activity. Login to comment 112 ABCC7 p.Gln493Ala ABCC7 p.Gln493Ala Because ATP binding may open the CFTR Cl-channel (13, 14), thereby reducing the interburst interval, we asked if the prolonged interburst interval in Q493A was consistent with attenuated binding. Login to comment 114 ABCC7 p.Gln493Ala Wild type CFTR and CFTR-Q493A had the same apparent EC50 for ATP (Fig. 3). Login to comment 121 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala Wild type CFTR, CFTR-Q493A, and CFTR-Q1291A were all inhibited by ADP. Login to comment 122 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala A, examples of patches incubated with ATP (1 mM) and ADP (1 mM) during the times indicated. B, data from multiple patches show that wild type CFTR, CFTR-Q493A, and CFTR-Q1291A currents were inhibited by ADP to a similar extent. Login to comment 125 ABCC7 p.Gln1291Ala CFTR-Q1291A was stimulated only weakly by PPi. Login to comment 127 ABCC7 p.Gln493Ala ABCC7 p.Gln1291Ala The membrane potential was clamped at -40 mV. Patches were incubated with 1 mM ATP and 4 mM PPi during the times indicated. B, data from multiple patches show that 4 mM PPi stimulated less Cl- current in CFTR-Q1291A (n ϭ 6) than in wild type CFTR (n ϭ 4) or CFTR-Q493A (n ϭ 4). Login to comment 130 ABCC7 p.Gln493Ala Fig. 4 shows that varying the divalent cation generated similar effects for wild type CFTR and the Q493A variant. Login to comment 134 ABCC7 p.Gln493Ala The Q493A mutation did not alter the response to either agent (Figs. Login to comment 136 ABCC7 p.Gln493Ala ABCC7 p.Asn505Cys Mutating Asn-505 in NBD1 Can Increase Channel Opening-The N505C mutation had an effect opposite to that of Q493A; it reduced the interburst interval and increased Po (Fig. 7). Login to comment 137 ABCC7 p.Gln493Ala As with Q493A, burst duration did not change. Login to comment 138 ABCC7 p.Asn505Ala CFTR-N505A had gating kinetics similar to those of wild type; presumably the introduced Ala did not disrupt the structure to the same extent as when Cys was substituted for Asn. Login to comment 140 ABCC7 p.Asn505Cys ABCC7 p.Asn505Ala Inhibition by ADP, stimulation by PPi, and the EC50 for ATP stimulation were not altered by the N505C or N505A mutations (data not shown). Login to comment 141 ABCC7 p.Gln493Ala ABCC7 p.Asn505Cys The opposite effects of the Q493A and N505C mutations on gating are consistent with the expectation that these mutations might have different effects on the ␥-phosphate linker. Login to comment 142 ABCC7 p.Gln1291Ala Mutating Gln-1291 in NBD2 Reduced PPi Stimulation of CFTR Channel Activity-In contrast to the NBD1 Gln mutant, the NBD2 Gln variant Q1291A showed gating much like wild type (Fig. 2). Login to comment 145 ABCC7 p.Lys1250Ala For example, the CFTR-K1250A mutant markedly prolongs burst duration (9, 10, 13, 22). Login to comment 146 ABCC7 p.Gln1291Ala Thus the normal burst duration in Q1291A suggests that mutation of the conserved Gln did not disrupt hydrolysis. Login to comment 147 ABCC7 p.Gln1291Ala Wild type CFTR and CFTR-Q1291A had the same apparent EC50 for ATP (Fig. 3) and the same response to variation of the divalent cation (Fig. 4). Login to comment 157 ABCC7 p.Gln1291Ala Despite the lack of effect of the Q1291A mutation on other aspects of gating, it dramatically reduced PPi stimulation (Fig. 6). Login to comment 160 ABCC7 p.Asn1303Lys The single-channel gating of CFTR-N1303K, a relatively frequent CF-associated mutation, showed a long burst duration and a long interburst interval (Fig. 8). Login to comment 161 ABCC7 p.Asn1303His ABCC7 p.Asn1303Ile ABCC7 p.Asn1303Ala We observed similar kinetic effects when Asn-1303 was changed to another CF-associated mutation (N1303H), a sequenced mutation with undetermined clinical consequences (N1303I), and an Ala (N1303A) (Fig. 8). Login to comment 167 ABCC7 p.Asn505Cys ABCC7 p.Asn505Ala B, data from multiple patches. Asterisk indicates p Ͻ 0.05 compared with wild type CFTR; n ϭ 4 for CFTR-N505A and 6 for CFTR-N505C. Login to comment 168 ABCC7 p.Asn1303Lys ABCC7 p.Lys1250Ala between CFTR-N1303K and CFTR-K1250A, we examined the effect of PPi and ADP. Login to comment 169 ABCC7 p.Asn1303Lys The N1303K mutation prevented PPi-dependent stimulation of current and eliminated ADP-dependent current inhibition (Fig. 9). Login to comment 170 ABCC7 p.Asn1303Lys Pyrophosphate caused only a 26.9 Ϯ 18.6% (n ϭ 4) increase in current for the CFTR-N1303K channel, and ADP caused only a 3.2 Ϯ 6.8% (n ϭ 3) inhibition in current. Login to comment 171 ABCC7 p.Asn1303Ala However, in contrast to the reduced EC50 for ATP observed for mutations at Lys-1250 (21), the apparent EC50 for CFTR-N1303A (253 Ϯ 62 ␮M, n ϭ 5) was not different from that of wild type CFTR (176 Ϯ 67 ␮M, n ϭ 5). Login to comment 180 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303His ABCC7 p.Asn1303Ile ABCC7 p.Asn1303Ala Comparison of single-channel kinetics of CFTR-N1303K, CFTR-N1303H, CFTR-N1303I, and CFTR-N1303A. Login to comment 184 ABCC7 p.Asn1303Lys Pyrophosphate and ADP did not affect CFTR-N1303K. Login to comment 187 ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys Addition of 4 mM PPi increased the burst duration of wild type CFTR but did not alter the burst duration of CFTR-N1303K. The addition of 1 mM ADP increased the interburst interval of CFTR but did not affect CFTR-N1303K. with mutation of the NBD1 Walker A lysine (9, 10, 22, 35). Login to comment 207 ABCC7 p.Asn1303Lys N1303K is a frequent CF-associated mutation that has been shown to affect channel processing (42). Login to comment 209 ABCC7 p.Asn1303His ABCC7 p.Asn1303Ile N1303H and N1303I also alter channel gating although their effect on Po was minor. Login to comment 211 ABCC7 p.Gln1291His ABCC7 p.Gln493Arg ABCC7 p.Gln1291Arg Two relatively uncommon missense mutations have been described at Gln-1291, Q1291H and Q1291R (43),4 and a single chromosome was reported to encode a variation at Gln-493, Q493R.4 The relatively mild gating abnormalities we observed for these variants suggest that if they cause CF, altered processing or some other abnormality may be the cause for the loss of CFTR function. Login to comment