Severed Channels Probe Regulation of Gating of Cystic Fibrosis Transmembrane Conductance Regulator by Its Cytoplasmic Domains

Abstract

Opening and closing of a CFTR Cl− channel is controlled by PKA-mediated phosphorylation of its cytoplasmic regulatory (R) domain and by ATP binding, and likely hydrolysis, at its two nucleotide binding domains. Functional interactions between the R domain and the two nucleotide binding domains were probed by characterizing the gating of severed CFTR channels expressed in Xenopus oocytes. Expression levels were assessed using measurements of oocyte conductance, and detailed functional characteristics of the channels were extracted from kinetic analyses of macroscopic current relaxations and of single-channel gating events in membrane patches excised from the oocytes. The kinetic behavior of wild-type (WT) CFTR channels was compared with that of split CFTR channels bearing a single cut (between residues 633 and 634) just before the R domain, of split channels with a single cut (between residues 835 and 837) just after the R domain, and of split channels from which the entire R domain (residues 634–836) between those two cut sites was omitted. The channels cut before the R domain had characteristics almost identical 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 domain were characterized by a low level of activity even without phosphorylation, strong stimulation by PKA, enhanced apparent affinity for ATP as assayed by open probability, and a somewhat destabilized binding site for the locking action of the nonhydrolyzable ATP analog AMPPNP. Split channels with no R domain (from coexpression of CFTR segments 1–633 and 837–1480) were highly active without phosphorylation, but otherwise displayed the characteristics of channels cut after the R domain, including higher apparent ATP affinity, and less tight binding of AMPPNP at the locking site, than for WT. Intriguingly, severed channels with no R domain were still noticeably stimulated by PKA, implying that activation of WT CFTR by PKA likely also includes some component unrelated to the R domain. As the maximal opening rates were the same for WT channels and split channels with no R domain, it seems that the phosphorylated R domain does not stimulate opening of CFTR channels; rather, the dephosphorylated R domain inhibits them.

Inhibition of CFTR Cl − channel function caused by enzymatic hydrolysis of sphingomyelin

“…7 E and F). This finding is consistent with the idea that the open state of CFTR-⌬R expressed in an oocyte is not as stable as that of the wild-type channel ''fully'' activated by PKA (9).…”

“…7B). To test the likely possibility that this variability reflects different cAMP levels in individual oocytes, we further boosted the cAMP level with the combination of 1 mM IBMX and 50 M forskolin (an adenylate cyclase stimulator) to maximally activate the CFTR channel (9). Under this condition, SMase C (which removes both choline and phosphoryl groups) inhibited 75% of the current (Fig.…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…Channel opening rate in an excised membrane patch is increased by adding PKA catalytic subunit in the presence of MgATP (Mathews et al 1998;Wang et al 2000). Burst duration also increases under highly phosphorylating conditions which implies that one or more PKA sites modulates channel closing (Csanády et al 2000). Most of the important PKA sites reside within the large R domain between NBD1 and TM7 (Fig.…”

“…Rich et al (1991) discovered that channels that lacked large portions of the R domain were more active in the absence of PKA stimulation. Csanády et al (2000) subsequently showed that split CFTR molecules that lack the R domain and distal NBD1 (missing residues 634 -836) formed channels with opening rates in the absence of PKA that approached maximal opening rates for fully phosphorylated wild-type channels. These findings confirm that channel opening is strongly inhibited by the unphosphorylated R domain ( perhaps with assistance from distal NBD1).…”

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

“…Substitutions at these residues result in increased channel conductance 10 , and removal of residues 760-783 or 817-838 produces active channels that open independently of phosphorylation 12,13 . Coexpression of CFTR and CFTR 837-1480 also produces low levels of constitutive Cl -channel activity, which is further stimulated with PKA 14 . The R region may have an additional stimulatory role, as shown by CFTR channels lacking much of the R region (Δ708-835/S660A), which gate independently of PKA yet are further stimulated by the addition in trans of phosphorylated R region (residues 645-835) 15 .…”