Severed channels probe regulation of gating of cystic fibrosis transmembrane conductance regulator by its cytoplasmic domains

Opening and closing of a CFTR Cl- channel is controlled by PKA-mediated pho sphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, a nd likely hydrolysis, at its two nucleotide binding domains. Functional int eractions between the R domain and the two nucleotide binding domains were probed by character -izing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of ooc yte conductance, and detailed functional characteristics of the channels we re extracted from kinetic analyses of macroscopic current relaxations and o f single-channel gating events in membrane patches excised from the oocytes . The kinetic behavior of wild-type (WT) CFTR channels was compared with th at of split CFTR channels bearing a single cut (between residues 633 and 63 4) just before the R domain, of split channels with a single cut (between r esidues 835 and 837) just after the R domain, and of split channels from wh ich the entire R domain (residues 634-836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost id entical to those of WT channels, except for less than twofold shorter open burst durations in the presence of PKA. Channels cut just after the R domai n were characterized by a low level of activity even without phosphorylatio n, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the lock ing action of the nonhydrolyzable ATP analog AMP- PNP. Split channels with no R domain (fr-om coexpression of CFTR segments 1-633 and 837-1480) were h ighly active without phosphorylation, but otherwise displayed the character istics of channels cut after the R domain, including higher apparent ATP af finity and less tight binding of AMPPNP at the locking site, than for WT. I ntriguingly, severed channels with no R domain were still noticeably stimul ated by PKA, implying that activation of WT CFTR by PKA likely also include s some component unrelated to the R domain. As the maximal opening rates we re the same for WT channels and split channels with no R domain, it seems t hat the phosphorylated R domain does not stimulate opening of CFTR channels ; rather, the dephosphorylated R domain inhibits them.