The long-term objectives are to understand the function and structure of inward rectifier K channels (IRKs). IRKs are important in excitable tissues such as nerve and muscle and our particular emphasis is on heart muscle. Here IRKs regulate resting membrane potential and the terminal phase of cardiac repolarization. Consequently their function is of great importance in evaluation and discovery of cardiac antiarrhythmic drugs, especially Class III antiarrhythmics. Three major hypotheses are examined: first whether specific naturally occurring cytoplasmic polyamines (PAs) regulate IRK function in cardiomyocytes; second whether the present topological model of IRKs is correct; and third whether a human cardiac IRK, hIRK is a major component of I-Kr, the rapid part of I-K the delayed rectifier K+ current of cardiomyocytes.
The specific aims are to: 1) localize C-terminus binding sites for both Mg2+ and PAs which together confer the unique property of inward rectification; 2) compare the effective valence and time-dependence of PA block with so-called """"""""intrinsic"""""""" gating of IRKs; 3) test the relationship between PA levels and IRK activity in cardiomyocytes; 4) est specific topological models of IRKs using glycosylation site insertion mutagenesis; and 5) identify the binding site for the Class Ill antiarrhythmic dofetilide, on hIRK and clone accessory subunit modifiers of this channel.
For Aims 1 and 2 and the first part of Aim 5, the research design uses methods of mutagenesis, heterologous expression and electrophysiology in an iterative manner.
For Aim 2 chemical interruption of PA metabolism is used to manipulate PA levels of cardiomyocytes while testing IRK function electrophysiologically.
For Aim 4 a genetically engineered IRK protein is overexpressed in a baculovirus-Sf9 system, immunopurified and tested for N-glycosylation at sites predicted to be extracellular by present topological models. Functional tests using patch clamp are performed simultaneously. For the second part of Aim 5, a variety of candidate modifier subunits that we have cloned will be tested for their ability to make hIRK more closely mimic I-Kr.
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