MOLECULAR ANALYSIS OF NEURONAL T TYPE CALCIUM CHANNELS

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DESCRIPTION: (Applicant's Abstract) Intracellular calcium controls a variety of cellular functions such as contraction, secretion, proliferation, and gene expression. One of the major pathways of calcium influx into cells is through voltage-activated Ca2+ channels. Low voltage-activated, T-type, Ca2+ channels can open after small depolarizations of the plasma membrane, leading to further depolarization of the membrane (pacemaker activity) and changes in intracellular Ca2+. T channels are also thought to play important roles in burst firing and in oscillatory behavior of neurons. Their ability to inactivate and recover quickly over a narrow voltage range are considered key properties of thalamic neurons, allowing them to show distinct firing patterns that correlate with sleep and wakefulness. Many antiepileptic drugs can block these channels in vitro, leading to the hypothesis that abnormal expression of T channels may be involved in epilepsy. Exciting preliminary studies demonstrate the cloning and expression of a new family of alpha1 subunits that encode T-type Ca2+ channels. Cloning of these channels has opened up new areas of research that should identify the physiology of this important class of ion channel. The specific aims of this project are to: 1) characterize the electrophysiological properties of these cloned T-type channels; 2) characterize their pharmacology, in particular their block by divalent cations, antihypertensives, and antiepileptics; 3) investigate structure-function relationships of T-type channels, focusing on inactivation properties; and 4) investigate the subunit structure of these channels, focusing on how Ca channels are regulated by their beta subunits. The research design uses recombinant DNA techniques to clone and modify T-type channels and express the cloned channels in both Xenopus laevis oocytes and HEK-293 cells. Electrophysiological methods are used to study the expressed channel at both the single channel and whole cell level. These studies should provide significant insights into the functional diversity of voltage-activated Ca channels, their pharmacology, and how their structure determines their function in neuronal signaling.