Function of Xenopus Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channels and Use of Human-Xenopus Chimeras to Investigate the Pore Properties of CFTR

doi:10.1074/jbc.271.41.25184

Function of Xenopus Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl⁻ Channels and Use of Human-Xenopus Chimeras to Investigate the Pore Properties of CFTR*

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

^∥ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed:
Howard Hughes Medical Institute, 500 EMRB, University of Iowa College of Medicine, Iowa City, IA 52242
. Tel.: 319-335-7619; Fax: 319-335-7623; E-mail: mjwelsh{at}blue.weeg.uiowa.edu

↵^¶ Current address: Depts. of Medicine and Biochemistry, University of Edinburgh, Edinburgh, United Kingdom.

Abstract

To explore the relationship between structure and function in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, we studied Xenopus CFTR. We found that the anion permeability sequence of cAMP-activated Cl⁻ currents in the apical membrane of Xenopus A6 epithelia differed from that of cAMP-activated Cl⁻ currents in human epithelia expressing CFTR. To understand the molecular basis for this difference and to learn whether CFTR from another species would have properties similar to human CFTR, we assembled a full-length Xenopus CFTR cDNA from A6 cells. Expression of Xenopus CFTR in HeLa cells generated cAMP-activated whole-cell currents and cAMP-dependent protein kinase-activated single channels that resembled those of human CFTR with the exception that the anion permeability sequence was different (Br⁻ = I⁻ > Cl⁻ in Xenopus CFTR and Br⁻ = Cl⁻ > I⁻ in human). In addition, the single-channel conductance of Xenopus CFTR was increased. To investigate protein regions that account for these differences, we constructed chimeric proteins by replacing either the first or second membrane-spanning domain of human CFTR with the equivalent region of Xenopus CFTR (hX1-6 and hX7-12, respectively) and examined their function in HeLa cells. We found that the anion permeability sequence (Br⁻ = I⁻ > Cl⁻) and single-channel conductance of hX1-6 resembled that of Xenopus CFTR expressed in HeLa cells, whereas hX7-12 had properties like those of human CFTR. However, the gating of hX1-6 showed a flickery behavior. The altered gating of hX1-6 was attributed to residues in the first extracellular loop of Xenopus CFTR because mutation of residues in that region to the corresponding residues of human CFTR produced gating behavior similar to that of human CFTR. These data suggest that sequence differences in the first membrane-spanning domains are responsible for the differences in the permeation properties of human and Xenopus CFTR and that the first extracellular loop influences channel gating.

Footnotes

↵^‡ These authors made equal contributions to this work.
↵^§ Associate of the Howard Hughes Medical Institute.
↵* This work was supported by the Howard Hughes Medical Institute, NHLBI, National Institutes of Health, and the Cystic Fibrosis Foundation. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U60209[GenBank].
↵¹ The abbreviations used are:

CFTR

cystic fibrosis transmembrane conductance regulator

MSD

membrane-spanning domain

NBD

nucleotide-binding domain

PCR

polymerase chain reaction

PKA

cAMP-dependent protein kinase

P_o

single-channel open-state probability

TES

(N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid)

pS

picosiemen(s).
↵² CFTR Cl⁻ currents exhibit a rectifying I-V relationship when the intracellular Cl⁻ concentration is reduced (15, 33). However, expression of hCFTR and XCFTR in HeLa cells generated cAMP-stimulated Cl⁻ currents that showed only weak rectification under the asymmetrical Cl⁻ concentrations used. The reason for the minimal outward rectification is not certain however, it may in part be caused by a loss of voltage-clamp within cells expressing high levels of recombinant CFTR. This would result in a significantly smaller transmembrane voltage than the applied voltage when currents are large, and hence currents would be relatively linear rather than outwardly rectifying.
↵³ Fig. 3 suggests that expression of hCFTR and XCFTR in HeLa cells generates cAMP-stimulated Cl⁻ currents with high selectivity for Cl⁻ (hCFTR E_rev ∼ −32 mV XCFTR E_rev ∼30 mV E_Cl = 32 mV). These data suggest greater selectivity than we reported previously for hCFTR using a larger Cl⁻ concentration gradient (15) but similar selectivity to what we reported previously when we used identical conditions (29). The reason for these differences in P_Na/P_Cl values is not known.
- Received March 11, 1996.
- Revision received June 18, 1996.