Catherine Proenza, PhD - US grants

Hyperpolarization-activated "pacemaker" currents are important for rhythmic firing in the mammalian heart and brain. This proposal addresses two previously undescribed features of pacemaker currents, (1) a voltage-independent instantaneous current, and (2) inactivation of mammalian currents. The molecular mechanisms responsible for these two current properties will be probed experimentally in heterologously- expressed pacemaker channels by a combination of molecular biology, chemical modification, and patch clamp electrophysiology. The presence of an instantaneous current would suggest that a background current accompanies expression of pacemaker channels in the heart and brain, and would raise the question of whether similar mechanisms exist in other ion channels. The hypotheses that cAMP-dependent inactivation existing in mammalian pacemarker currents and is altered by intracellular factors suggest a novel mechanism for modulation of these currents. Determination of the molecular mechanism for an instantaneous current and/or inactivation of mammalian pacemaker currents would advance our understanding of the control and modulation of spontaneous rhythmic activity in the heart and brain. Description of these channel behavior may also present opportunities for development of state-dependent pharmacological agents that act on pacemaker channels.

The long term goals of this project are to understand the molecular and biophysical mechanisms for the regulation of cardiac pacemaking in sinoatrial node myocytes (SAMs) across the gamut of physiological conditions. SAMs function as cardiac pacemaker cells by firing spontaneous action potentials (APs). As in other excitable cells, the precise shape of sinoatrial APs reflects the composite activity of the unique complement of ion channels and transporters on the plasma membrane. AP waveforms are not static; they vary in response to short- and long-term changes in physiological context. In principle, differences in AP waveforms should lend insight into the changes in ionic currents that underlie cellular electrophysiological responses. However, our ability to decode the causal relationships between ionic currents and AP shape remains an elusive goal in all excitable cells. This gap in understanding is caused by a lack of information about AP waveforms and currents in different physiological contexts and by difficulties inherent to the study of interrelated systems using conventional research approaches. The present proposal addresses these general questions by focusing on the mechanisms by which aging slows cardiac pacemaking. Proposed experiments follow from work in prior funding periods and new preliminary data which show that aging slows pacemaking in part by decreasing the spontaneous AP firing rate of SAMs in association with changes in a limited subset of AP waveform parameters and reductions in the funny current (If) and voltage-gated Ca2+ currents (ICa,L and ICa,T). They also address the prior observation that age-dependent reductions in pacemaker activity and If in SAMs can be reversed by high concentrations of exogenous cAMP via a cAMP-mimetic mechanism. Proposed experiments will use new research tools developed during the current funding period (1) to define age-dependent changes in the relative contributions of currents active during different phases of the AP in SAMs, (2) to test the ability of different currents, singly and in combination, to transform the AP phenotype of young and aged SAMs, and (3) to test the hypothesis that age-dependent reduction in a novel If regulatory protein is responsible for the hyperpolarizing shift in voltage-dependence and resulting slowing of AP firing rate in SAMs and heart rate in mice. Results of these studies will experimentally define for the first time causal links between individual ionic currents and AP waveform parameters in SAMs that are responsible for cardiac pacemaking in general and will reveal how these mechanisms are changed during normal aging.