The long term goal is a deeper understanding of the cellular mechanisms that regulate cardiac """"""""background"""""""" currents which, in turn, control diastolic potentials, influence automaticity, affect the amplitude and duration of the action potential plateau, and play a role in the initiation and termination of cardiac arrhythmias. Present focus is on regulation of a time- and voltage-independent background C1 conductance, activated via cAMP-dependent-protein-kinase (PKA)-mediated phosphorylation, in guinea pig ventricular myocytes. The underlying cardiac C1 channels resemble in several respects the airway epithelial C1 channels (called CFTR: cystic fibrosis transmembrane conductance regulator) whose phosphorylation by PKA appears defective in cystic fibrosis patients, and our preliminary Northern blot suggest that CFTR is expressed in guinea pig ventricle. Phosphorylation is necessary, but not sufficient, to open these cardiac C1 channels which, like CFTR, require in addition a hydrolyzable nucleoside triphosphate. Because the C1 equilibrium potential in cardiac cells lies just positive to the resting potential, during sympathetic or histaminergic stimulation a substantial outward C1 current should flow throughout the action potential plateau, accelerating action potential repolarization and, hence, safeguarding impulse propagation in the face of possibly arrhythmogenic influences of the simultaneously enhanced Ca channel current and increased heart rate. The specific objectives are to continue characterization of the cardiac PKA-activated C1 channel and of the mechanisms of its modulation, and to determine whether it is indeed identical to CFTR. Two related experimental approaches are used, both of which permit access to the cytoplasmic face of the sarcolemma during current recording. The first is the whole-cell version of the patch-clamp technique in which myocytes are voltage clamped and internally dialyzed via wide tipped (about 5 micro m), low resistance (about 1 Momega), patch pipettes fitted with a simple device for exchanging the solution inside the pipette tip, and hence inside the cell, during current recording. With this approach, agents such as receptor agonists or antagonists, or forskolin, can be rapidly applied to the cell exterior, and agents added to pipette solutions, such as ATP, cAMP, GTP, or their hydrolysis-resistant analogs, or specific inhibitors of kinases or phosphatases, can diffuse readily into the cell interior. The second approach is the novel excised """"""""giant"""""""" patch technique using pipettes with tip diameters greater than 15 micro m to isolate large inside-out patches of sarcolemmal membrane. This affords direct, unhindered, and rapid access to the cytoplasmic face of the sarcolemma of not only nucleotides, and peptide and other inhibitors, but also much larger molecules such as purified kinases and phosphatases. Kinetic analysis of single-channel currents from these patches over a wide range of experimental conditions will allow determination of the channel's gating mechanisms which, in turn, should provide molecular insight to aid the rational design of clinical interventions (directed at cardiac and/or epithelial CF abnormalities).
| 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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