Members

Ion channels are proteins that form ion-selective pores within cell membranes. In the heart, ion channels establish and maintain normal cardiac rate and rhythm, and changes in their function due to genetic or environmental causes can lead to life-threatening alterations of the cardiac rhythm. Despite such a critical role, the molecular mechanisms underlying ion channel function and how these are modulated by cardiovascular disease are unclear. In our research, we use fluorescence-based electrophysiology approaches to study ion channel behaviour. These cutting-edge techniques provide a novel and innovative tool to study the relationship between ion channel dynamic structure and its function. They allow us to directly observe changes in channel protein structure that underlie normal function and to visualize the effects of polymorphisms and environmental changes that are associated with cardiovascular disease. This gives us a new and powerful way to study ion channel biophysics and physiology.

The Paetzel Lab

Our lab uses molecular biology, protein chemistry, and X-ray crystallography to investigate the structure and mechanism of the proteins and protein complexes involved in targeting, translocation and catalysis at the cellular membrane surface. We are particularly interested in membrane bound proteases.

The long-term goals of our research are to assess the biophysical sequelae of identifiable sodium channel mutations and substitutions that lead to changes in cellular excitability and toxin resistance. The specific experimental aims of our research are:

1. to explore the molecular determinants and biophysical underpinnings of diseases of excitability in cardiac muscle, skeletal muscle, and neurons;
2. to use toxin resistance in sodium channels as a marker for adaptation and parallel evolution;
3. to determine the interactions between activation, deactivation, fast inactivation and slow inactivation in the regulation of sodium channel availability and the contribution of sodium channels to cell excitability, the responsiveness of cells to excitatory synaptic input, and the productionof action potentials.

The research program in the Cardiac Membrane Research Lab (CMRL) focuses on the cellular and molecular mechanisms by which the heart adapts to physiological, pathological and environmental perturbations. The strength of cardiac muscle contraction is regulated by the cytosolic Ca2+ activity ([Ca2+]i) on a beat-to-beat basis. We focus, therefore, on factors which control [Ca2+]i in the heart. These studies are multidisciplinary and conducted at three different levels of organization. The first and most integrative approach makes use of isolated single cardiac cells in which speed of shortening is assessed by video microscopy using edge detection and the [Ca2+]i transient is determined on line with indo-1 fluorescence microscopy. The second approach involves measuring Ca2+ movement across isolated membranes using voltage clamp, planar lipid bilayers and/or radioisotopic techniques. The third approach includes the cloning, sequencing and expression of proteins critically involved in Ca2+ handling. These proteins include the: L-type voltage-dependent Ca2+ channel (DHPR), which controls Ca2+ influx; Na+/Ca2+ exchanger (NCX), which is the primary mechanism of Ca2+ efflux; and troponin C, a Ca2+ binding protein which is crucial in the initiation of contraction. These proteins and mutants produced by site-directed mutagenesis are studied in vitro as well as in the reconstituted muscle fibre. With these techniques, we are investigating the mechanisms by which these proteins are regulated in different species to meet a variety of physiological, pathological and environmental demands.

Cyclic Nucleotide Regulation of Pacemaker HCN Channels

Among the many ion channels controlling electrical signaling in the heart, the "pacemaker" HCN ion channel plays a special role by responding simultaneously to membrane voltage and to chemical messenger molecules, namely cyclic nucleotides. We are studying how binding of cyclic nucleotides to the HCN channel causes distinctive structural changes in the channel protein, with complex functional consequences. Our lab employs a combination of electrophysiological and biochemical techniques to study this multifaceted ion channel, as well as related proteins drawn from other physiological contexts.