The overall goal of the research in this laboratory is to provide an understanding of the mechanisms involved in the regulation and modulation of myocardial membrane excitability. To achieve this goal, it is necessary to characterize the properties of the various ionic conductance pathways present in myocardial cells and to determine how these conductance pathways are affected by neurotransmitters, neurohormones and intracellular second messengers. This proposal is specifically focused on characterizing the properties of depolarization-activated K+ currents in isolated adult rat ventricular and atrial myocytes and on delineating the mechanisms involved in mediating the effects of ventricular and atrial myocytes and on delineating the mechanisms involved in mediating the effects of alpha1- adrenergic receptor stimulation on these currents. Using the whole-cell variation of the patch clamp recording technique, initial experiments will determine the ionic selectivities of the depolarization-activated currents, Ito and IK, ventricular myocytes. In parallel studies, the time-and voltage-dependent properties and the ionic selectivity of the K+ currents in atrial cells will be characterized. To study the kinetics and dose- dependences of alpha1-receptor mediated attenuation of Ito and IK in ventricular cells, a photoactivatable analogue of norepinephrine (""""""""caged- NE"""""""") will be developed and used. The effects of concentration """"""""jumps"""""""" of NE, produced by light flashes in the presence of """"""""caged-NE"""""""", on Ito and IK will be quantified and compared. The effects of alpha1-receptor stimulation on K+ currents in atrial cells will also be examined using """"""""caged-NE"""""""". Mechanistic studies will focus on delineating the intracellular pathways involved in mediating and transducing the effects alpha1-adrenergic receptor stimulation on outward K+ currents in ventricular myocytes. These studies will determine the roles of protein kinase C (PKC), cAMP hydrolysis and IP3 in mediating the effects of alpha1- agonists and roles of GTP and GTP-binding proteins in transducing the effects of alpha1-adrenergic receptor stimulation on K+ currents in these cells. In these experiments, intracellular perfusion and light flash techniques will be employed to manipulate the activities or the concentrations of PKC, cAMP, IP3 and GTP in cells during whole-cell recordings. It is unlikely that any direct clinical applications result will from the studies proposed here. It is expected, however, that these studies will better define the physiological role of depolarization-activated K+ channels in the regulation and modulation of myocardial membrane excitability. In addition, it is anticipated that these studies will provide important insights into the role of alpha1-adrenergic receptor stimulation in the autonomic regulation of cardiac function and into the mechanism involved in mediating these effects.
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