A long term goal is a detailed understanding of the many cellular mechanisms that modulate cardiac """"""""background"""""""" membrane currents, which can contribute to diastolic potentials, to the plateau of the cardiac action potential, and to the initiation and termination of cardiac arrhythmias. Sharp focus is presently on cardiac CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl channels which, functionally, seem closely similar to the epithelial CFTR Cl channels that are dysfunctional (often due to defective processing) in cystic fibrosis patients.
The specific aims are (1) to pursue detailed characterization of the endogenous cellular regulatory mechanisms of mammalian cardiac CFTR Cl conductance in intact myocytes, the (2) to determine the gating mechanisms of individual cardiac CFTR channels in excised patches of myocyte membrane. Cardiac myocytes are presently one of the few (if not the only) preparations in which it is possible to examine both the complex regulation of native CFTR channels in their natural physiological environment by endogenous kinase and phosphatase systems, and the underlying gating behavior of single channels. The work will allow a rigorous comparison between the functional properties of epithelial and cardiac CFTR channels. Two related experimental approaches are used: whole-cell current recording in intact myocytes that are voltage clamped and internally dialyzed via wide tipped patch pipettes fitted with a pipette perfusion device, via which nucleotides or their analogs, or specific inhibitors of kinases of phosphatases, can be readily applied to the cell interior; and the excised """"""""giant"""""""" patch technique for recording unitary channel currents in large inside-out patches of sarcolemmal membrane, to the cytoplasmic face of which nucleotides, peptide inhibitors, and also much larger molecules such as purified kinases and phosphatases, can be directly and rapidly applied. Kinetic analyses of single-channel currents from these patches will advance understanding of the channel's gating mechanisms. It appears that, during each open-close gating cycle, a single highly-phosphorylated CFTR channel hydrolyzes one molecule of ATP at its N-terminal nucleotide binding domain to open, and then hydrolyzes a second ATP at its N-terminal nucleotide binding domain to close. CFTR channels thus offer an opportunity, unprecedented in biology, to examine individual ATP hydrolysis cycles in a single protein molecule, in its natural environment, in real time.
| Gadsby, David C; Takeuchi, Ayako; Artigas, Pablo et al. (2009) Review. Peering into an ATPase ion pump with single-channel recordings. Philos Trans R Soc Lond B Biol Sci 364:229-38 |
| Gadsby, David C (2009) Ion channels versus ion pumps: the principal difference, in principle. Nat Rev Mol Cell Biol 10:344-52 |
| Dousmanis, Athanasios G; Nairn, Angus C; Gadsby, David C (2002) Distinct Mg(2+)-dependent steps rate limit opening and closing of a single CFTR Cl(-) channel. J Gen Physiol 119:545-59 |
| Csanady, L; Gadsby, D C (1999) CFTR channel gating: incremental progress in irreversible steps. J Gen Physiol 114:49-53 |
| Gadsby, D C; Nairn, A C (1999) Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol Rev 79:S77-S107 |
| Gadsby, D C; Nairn, A C (1999) Regulation of CFTR Cl- ion channels by phosphorylation and dephosphorylation. Adv Second Messenger Phosphoprotein Res 33:79-106 |
| Gadsby, D C; Dousmanis, A G; Nairn, A C (1998) ATP hydrolysis cycles and the gating of CFTR Cl- channels. Acta Physiol Scand Suppl 643:247-56 |
| Senior, A E; Gadsby, D C (1997) ATP hydrolysis cycles and mechanism in P-glycoprotein and CFTR. Semin Cancer Biol 8:143-50 |
| Gadsby, D C; Nagel, G; Hwang, T C (1995) The CFTR chloride channel of mammalian heart. Annu Rev Physiol 57:387-416 |
| Hwang, T C; Nagel, G; Nairn, A C et al. (1994) Regulation of the gating of cystic fibrosis transmembrane conductance regulator C1 channels by phosphorylation and ATP hydrolysis. Proc Natl Acad Sci U S A 91:4698-702 |
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