Kanelis, Voula

Research

Investigating the Molecular Basis for Regulation of ABC Transporters

The ATP-binding cassette (ABC) superfamily of transporters are multidomain, integral membrane proteins found in all species. At minimum, these proteins consists of two repeats each comprised of a membrane spanning domain and a cytoplasmic nucleotide binding domain (NBD) (Figure 1). Intracellular loops extend from the transmembrane helices and link the NBDs and membrane domains. ATP binding induces the formation of an NBD dimer which dissociates upon ATP hydrolysis, with the associated conformational changes transmitted to the membrane spanning domains to allow ligand binding or solute transport. Although poorly understood at the molecular level, proper regulation of this process is essential. Many genetic diseases, including cystic fibrosis and cardiomyopathies, are caused by mutations in the NBDs. Therefore, studies of the NBDs and their interactions are critical to understanding the precise mechanism of ABC transporter regulation in normal states and their dysfunction in diseases.

Research in my laboratory will investigate the molecular basis of regulation of ABC transporters. As a model system, our studies will focus on the sulfonylurea receptor (SUR) family of ABC transporters which form the regulatory subunits of the ATP-sensitive K+ (KATP) channels necessary for proper pancreatic and cardiovascular function. Proper regulation of KATP channels by the SUR subunits is essential as mutations, particularly in the NBDs, cause a number of diseases, including type II diabetes mellitus, myocardial infarctions, and cardiomyopathies. Therefore, studies of the NBDs are essential in order to gain insights into the molecular basis of KATP channel regulation by the SUR proteins, which is impaired in prevalent human diseases. Further, information gleaned on regulation of SUR protein function will be applicable to other members of the ubiquitous ABC transporter family, and thus will shed light on the molecular basis of regulation in these proteins.

A combination of biochemistry, biophysical experiments, and NMR spectroscopy will be employed to investigate the role of the NBDs in regulation of SUR function. These experiments will probe protein-protein interactions, structure, and dynamics. The use of NMR spectroscopy will provide the residue-level resolution necessary for understanding the molecular mechanisms involved in SUR protein regulation of KATP channels. Our initial studies will be focused on wild type SUR proteins, with studies of disease-causing mutations comprising longer term objectives.

The specific goals of this research program are (i) to investigate interactions involving the SUR NBDs necessary for proper SUR protein structure and function, and KATP channel regulation, (ii) to obtain structural data on SUR NBD complexes integral to SUR-mediated KATP channel regulation, and (iii) to probe protein dynamics of isolated NBDs and NBD complexes involved in coupling ATP hydrolysis at the NBDs to solute transport at the membrane spanning domains.