Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

doi:10.1074/jbc.274.9.5429

Cystic Fibrosis-associated Mutations at Arginine 347 Alter the Pore Architecture of CFTR

EVIDENCE FOR DISRUPTION OF A SALT BRIDGE*

From the Howard Hughes Medical Institute and Departments of Internal Medicine and Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242

Abstract

Arginine 347 in the sixth transmembrane domain of cystic fibrosis transmembrane conductance regulator (CFTR) is a site of four cystic fibrosis-associated mutations. To better understand the function of Arg-347 and to learn how mutations at this site disrupt channel activity, we mutated Arg-347 to Asp, Cys, Glu, His, Leu, or Lys and examined single-channel function. Every Arg-347 mutation examined, except R347K, had a destabilizing effect on the pore, causing the channel to flutter between two conductance states. Chloride flow through the larger conductance state was similar to that of wild-type CFTR, suggesting that the residue at position 347 does not interact directly with permeating anions. We hypothesized that Arg-347 stabilizes the channel through an electrostatic interaction with an anionic residue in another transmembrane domain. To test this, we mutated anionic residues (Asp-924, Asp-993, and Glu-1104) to Arg in the context of either R347E or R347D mutations. Interestingly, the D924R mutation complemented R347D, yielding a channel that behaved like wild-type CFTR. These data suggest that Arg-347 plays an important structural role in CFTR, at least in part by forming a salt bridge with Asp-924; cystic fibrosis-associated mutations disrupt this interaction.

Footnotes

↵* This work was supported by Grants HL29851 and HL42385 from the NHLBI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 500 EMRB, Iowa City, Iowa 52242. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail:mjwelsh{at}blue.weeg.uiowa.edu.
↵2 C. Ferec, personal communication.
↵3 The shift toward a lower pK for mutants with more rapid kinetics (Fig. 1 and 3 B) may be a filtering artifact; from analysis of the R347E mutant and the R347H mutant, we know that the O_B state tends to be shorter then the O_L state at the applied transmembrane voltage and is therefore more vulnerable to filtering. We presume that this relationship is preserved throughout all the mutants.
Abbreviations:

CFTR

cystic fibrosis transmembrane conductance regulator

M

transmembrane domain

MSD

membrane-spanning domain

Bis-Tris

bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane

MOPS

3-(N-morpholino)propanesulfonic acid

TES

N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid

Tricine

N-tris(hydroxymethyl)methylglycine

DIDS

4,4′-diisothiocyanostilbene-2,2′-disulfonic acid

NMDG

N-methyl-d-glucamine

I-V

current-voltage relationship

pH_c

cytosolic, bath pH

pH_o

extracellular, pipette pH

O_L

little conductance state

O_B

big conductance state

τ_L

lifetime of little conductance state

τ_B

lifetime of big conductance state
- Received November 9, 1998.
- Revision received December 7, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.