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Use of human-murine CFTR chimeras to investigate the coupling of permeation and gating in the CFTR chloride channel

Reference	BB/C517517/1
Principal Investigator / Supervisor	Professor David Sheppard
Co-Investigators / Co-Supervisors	Dr A Boyd
Institution	University of Bristol
Department	Physiology and Pharmacology
Funding type	Research
Value (£)	276,936
Status	Completed
Type	Research Grant
Start date	01/05/2005
End date	30/04/2008
Duration	36 months

Abstract

The epithelial chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) transporter superfamily. CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a regulatory (R) domain. The MSDs form an anion-selective pore that is tightly controlled by phosphorylation of the R domain and ATP binding and hydrolysis at the interface of a dimmer formed by the two NBDs. In previous work, we demonstrated that human and murine CFTR exhibit marked differences in gating behaviour and pharmacology. To identify the protein regions responsible for these differences, we constructed a series of human-murine CFTR chimeras using recombinational cloning technology. We replaced NBD1, NBD2 and the R domain of human CFTR with the equivalent regions of murine CFTR to form the chimeras hmNBD1, hmNBD2 and hmRD, respectively. Using the patch-clamp technique, we demonstrated that NBD2 is a crucial determinant of the pharmacological differences between human and murine CFTR. However, because the gating behaviour of the chimeric channels resemble that of human CFTR, neither the NBDs nor the R domain account for the differences in gating behaviour between human and murine CFTR. We now propose to employ human-murine CFTR chimeras to investigate the coupling between anion permeation and channel gating in the CFTR chloride channel. We will begin by probing the biophysical properties and pharmacology of the murine CFTR pore and developing kinetic models of the gating behaviour of murine CFTR. Using permeant anions with different physical characteristics and a variety of open-channel blockers, we will quantify the permeation and conduction properties of murine CFTR and evaluate the architecture of the channel pore. Because of the anion selectivity of human CFTR is dynamic, we will pay special attention to how anion selectivity is controlled in murine CFTR. We will elucidate the molecular basis of this regulation by employing high-resolution single-channel recording. To identify the protein regions responsible for the differences in gating behaviour between human and murine CFTR, we will construct further chimeric proteins. Two attractive regions for study are the intra- and extracellular loops, which link the transmembrane segments of the MSDs. Concurrent with studies of the anion selectivity and gating of CFTR chimeras, we will investigate their biosynthesis and ATPase activity. By studying the biosynthesis of chimeric proteins, we will learn how different regions of CFTR determine protein folding and stability. Moreover, by analysing the ATPase function of chimeric CFTRs, we will gain novel insight into how the NBDs control anion flow through the CFTR pore. Taken together, our results will provide new understanding of CFTR structure and function. This knowledge is essential for the understanding of epithelial ion transport and the physiology of organs lined by epithelia. It might also provide new insight into how ATP hydrolysis drives substrate translocation in ABC transporters.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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