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

NEG2, a short C-terminal segment (817–838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.