ABCMdb

ABCC7 p.Ser768Cys

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Publications

PMID: 21059651 [PubMed] Wang G et al: "The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator."

No. Sentence Comment

91 RESULTS Disulfide Cross-linking of the R Domain to CL3-If Ser768 inhibits channel activity by interacting with outwardly facing residues from CL3, disulfide cross-linking of S768C to a corresponding cysteine inserted in CL3 will be expected to suppress channel activity. Login to comment
92 As a negative control, S768C cannot form an inhibitory disulfide bond with an inwardly facing cysteine mutated in CL3. Login to comment
94 S768C or S737C were fixed as an anchor point to search other inhibitory targets from CL3 (Fig. 2A). Login to comment
95 S768C/H950C was a representative example (Fig. 2B). Login to comment
99 B-D, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing mutants H950C/S768C (B), H950C (C), and S768C (D) by using a ramp protocol (Ϯ80 mV). Login to comment
108 F, unitary currents from a H950C/S768C construct in the presence of 20 ␮M diamide (b) and 4 mM DTT (c). Login to comment
115 In contrast, both diamide and DTT had no effect on H950C and S768C CFTR constructs (Fig. 2, C and D). Login to comment
116 These observations clearly suggest that a disulfide bond may be formed between S768C and H950C. Login to comment
118 Supporting this argument, internal diamide also inhibited activity of a mutant S768C/K951C, S768C/H954C, or S768C/ S955C, and inhibition was reversed by 4-6 mM DTT (Fig. 2E). Login to comment
121 However, S768C could not form an inhibitory disulfide bond with V956C (inwardly facing) or K946C, possibly as a result of a long distance or a poor relative orientation (Fig. 2E). Login to comment
125 Fig. 3 demonstrates that a CFTR construct with a single cysteine S768C, S737C, H950C, or H954C exhibited a clear single band no matter whether diamide or DTT was added. Login to comment
126 In sharp contrast, CFTR constructs with a cysteine pair (Cys-free background), S737C/H950C, S737C/H954C, S768C/H950C, and S768C/H954C, exhibited an additional cross-linked (X-linked) band because it was induced by diamide but was weakened by DTT. Login to comment
128 Therefore, a disulfide bond can be formed between H950C (or H954C) and S768C or between H954C (or H950C) and S737C. Login to comment
129 In order to address if the disulfide bond changes the gating kinetics, a two-channel recording of the H950C/S768C construct was done. Login to comment
159 In addition, because S768D is negatively charged, an electrostatic attraction between S768D and Lys946 or Lys951 may be impossible because modification of S768C with MTSCE (negatively charged) or MTSET (positively charged) failed to change channel activity (supplemental Fig. S1, A and B). Login to comment
162 Because disulfide cross-linking of S768C to H950C inhibited more channel activity than that of S768C to S955C (Fig. 2E), it is more possible for His950 to form an inhibitory H-bond with Ser768 . Login to comment
165 Supporting this proposal, modification of S768C with MTSEA (a very strong H-bond donor (33)) also inhibited 25% of channel activity after the channel was activated by ATP and PKA followed by PKI to block further phosphorylation (supplemental Fig. S1, C and D). Login to comment
244 Although thiol-specific disulfide cross-linking of S768C to H950C or nearby cysteines inserted in CL3 inhibited channel activity primarily by stopping the channel from opening, an electrostatic expulsion between S768R/D and H950R/D clearly promoted channel opening even in the absence of ATP. Login to comment
288 Second, diamide-induced disulfide bond cross-linking of S768C to H950C or its neighboring cysteines, which was confirmed by the SDS-PAGE mobility (Fig. 3), inhibited channel activity (Fig. 2). Login to comment
304 Error bars, S.E. Inhibition of CFTR by Ser768 2180 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 3•JANUARY of S768C with MTSEA (a strong H-bond donor (33)), but not with MTSCE (a strong H-bond acceptor), inhibited channel activity, whereas modification with MTSET failed to (supplemental Fig. S1). Login to comment

PMID: 23060444 [PubMed] Wang G et al: "Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3."

No. Sentence Comment

123 To support this hypothesis, we determined if S832C is closed enough to S768C to form a detectable disulfide-bond crosslinking based on the Cys-free CFTR construct. Login to comment
124 Fig. 7 demonstrates that 10M diamide greatly suppressed the channel activity of S768C/S832C/V510A mutant but failed to affect the channel activities of both S768C/V510A and S832C/V510A. Login to comment
154 Finally, D979A in CF patients causes misprocessing (33). Login to comment
153 To support this hypothesis, we determined whether S832C is closed enough to S768C to form a detectable disulfide bond cross-linking based on the Cys-free CFTR construct. Login to comment
195 Effects of diamide on CFTR mutants at the R-CL3 interface. A-C, macroscopic currents across inside-out membrane patches excised from transfected HEK-293T cells expressing CFTR mutants S768C/V510A (A), S832C/V510A (B), and S768C/S832C/V510A (C) by using a ramp protocol (afe;80 mV). Login to comment