Structure-Function Studies on Human Epithelial Cystic Fibrosis Chloride Channels Expressed in Xenopus Laevis Oocytes

<p>The cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel is an ATP-binding cassette protein, comprising two transmembrane domains, two nucleotide binding domains (NBD1, NBD2) and a regulatory (R) domain. Channel gating is controlled by R-domain phosphorylation and by ATP binding and/or hydroysis at the NBDs. Exon 13 of the CFTR gene encodes residues 590 to 830, originally ascribed to the R domain. Here, CFTR channels were severed near likely N- or C-terminal boundaries of NBD1 or the R domain. Channel activity, assayed via two-microelectrode voltage clamp monitored successful assembly of pairs of channel segments as the sever point was systematically shifted along the primary sequence. Substantial activity indicated successful assembly; such constructs were further studied in excised patches, by correlating macroscopic and single-channel kinetics. The C-terminus of NBD1 was found to extend beyond residues 590 and lies between residues 622 and 634, while the N-terminus of NBD1 lies between residues 432 and 449. In excised patches, channels severed just before (between a.a.s 432 and 433) or after NBD1 (between residues 633 and 634) displayed the usual hallmark characteristics of wild-type (WT) CFTR: requirement of phosphorylation by protein kinase A (PKA) and exposure to ATP for gating, ability to be locked open by pyrophosphate or AMPPNP, small single-channel conductances, and high apparent affinity of channel opening by although bursts of 1-633 plus 634-1480 channels were -40% shorter than in WT. Channels severed near the R domain's C-terminus, 1-835 plus 837-1480, displayed low, PKA-independent, activity, enhanced apparent affinity for ATP, and destabilized binding site for the locking action of AMPPNP. R-domain-less split channels 1-633 plus 837-1480 were functionally similar to 1-835 plus 837-1480, including enhanced apparent ATP affinity and less tight binding of AMPPNP, but were more active before phosphorylation. Intriguingly, 1-633 plus 837-1480 channels, lacking the R domain, were still stimulated by PKA; and AMPPNP or pyrophosphate still prolonged bursts. The kinetic analysis exploited a new method that rapidly extracts single-channel transition rate constants from multichannel patch-clamp recordings, using a simultaneous maximum-likelihood fit to the dwell-time distributions for all conductance levels (after successfully testing it on simulated current traces).</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

2000

Author dc:creator

• Csanady, Laszlo

Contributors dc:contributor

• David Gadsby

• CFTR
• ATP binding
• NBD1
• R domain
• phosphorylation
• channel gating
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/320

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1319