CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is a chloride channel that plays a critical role in secretion and absorption of water and electrolytes across epithelia. Since CFTR channels are also expressed in cardiac myocytes and are found to shorten action potential duration and induce repetitive activity, they are implicated to be arrythmogenic. One unique feature of the CFTR channel as an ion channel is that the free energy of ATP hydrolysis is harvested to drive the conformational changes that open and close the channel. Studies using mutant CFTR and various ATP or phosphate analogs have suggested a model that ATP hydrolysis at two nucleotide binding sites is tightly coupled to the opening and closing of the channel pore. Our understanding of the molecular basis of the coupling mechanism, however, remains primitive. Unresolved questions include: What is the stoichiometry of ATP binding/hydrolysis to gating transitions? How are the biochemical states in ATP hydrolysis cycles translated to the open and closed states in the gating transitions? Which part of the protein forms the aqueous pore? What is the relationship between the gate and the pore? These are fundamental questions that interest a broad spectrum of physiologists. A combinational approach is being adopted to tackle the molecular physiology of CFTR chloride channels. Different configurations of the patch-clamp techniques will be used to record CFTR channel activity so that both the cytoplasmic and the extracellular side of the channel are accessible to channel blockers, modifiers, or channel openers. Mutations in critical regions of the protein will be made to study the functional consequences of single amino acid substitutions on gating and permeation/blocking. State-dependent chemical modifications of engineered cysteines allow us to explore the dynamic protein conformational changes during gating transitions.
The specific aims of the project are:
Aim 1. To understand how ATP hydrolysis is coupled to the opening and closing transitions of CFTR.
Aim 2. To probe the CFTR pore with permeant and impermeant anions.
Aim 3, To study the structure-function relationship between the gate and the pore of CFTR. A clear understanding of the molecular mechanisms of CFTR function will aid in the design of pharmacological agents for therapeutic intervention in cystic fibrosis, secretory diarrhea and cardiac arrythmia.
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