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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2

Sealy Center for Structural Biology and Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0437

Submitted 23 January 2004 ; accepted in final form 21 July 2004

Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus (X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability (P_o) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance () was increased by 22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH₂-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in P_o (with a small change in ) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains.

cystic fibrosis; chloride channel; protein kinases; ATP binding cassette proteins

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