Muscarinic acetylcholine receptors (mAChRs) are a family of five structurally related subtypes (m1 - m5) which interact with guanine nucleotide binding (G) proteins to regulate a variety of important physiological responses. Within the central nervous system, mAChRs frequently modulate neuronal excitability through the suppression of several types of potassium channels. In addition, mAChRs negatively regulate heart contraction through the activation of a G protein-gated inward rectifier potassium channel. The proposed studies will provide important information regarding the biochemical mechanisms by which mAChR subtypes regulate cell excitability through the modulation of cloned potassium channels, therefore, these studies may identify particular targets for therapeutic intervention in diseases such as cardiac arrhythmia. We have recently shown that the m1 mAChR potently suppresses a cloned delayed rectifier potassium channel through a novel pathway involving receptor-stimulated tyrosine kinase activity. To better characterize this pathway, the tyrosine kinase responsible for channel suppression will be isolated through biochemical and immunological assays. The isolated kinase will then be tested for its effects on individual potassium channels. In addition, cell transfection studies with normal and mutant derivatives of the kinase will be conducted to determine the role of this kinase in vivo. In addition to testing the effect of this kinase on known ion channels, experiments will be conducted to determine whether the kinase is involved in processing of the Alzheimer Precursor Protein. To understand the G protein-dependent mechanism involved in activation of the cardiac inward rectifier channel, electrophysiological studies will be conducted to determine the role of individual G proteins in channel gating. In addition, a series of mutations will be introduced into the cloned channel to identify sites of G protein interaction. Furthermore, protein-protein interaction assays will be conducted to determine whether G protein subunits directly bind the inward rectifier channel. Since this represents the first cloned, G protein-activated channel, these studies may serve as a model for the investigation of related channels expressed within the brain and the periphery.
| Bard, J; Kunkel, M T; Peralta, E G (2000) Single channel studies of inward rectifier potassium channel regulation by muscarinic acetylcholine receptors. J Gen Physiol 116:645-52 |
