The long-term goal of this proposal is to study the role which neuronal voltage-gated Ca2+ channels play in chemical-induced toxicity, and disease processes. These channels regulate numerous critical cellular functions including secretion of neurotransmitters and hormones, regulation of excitability, condition of action potentials in cardiac and smooth muscle as well as in dendritic regions of some neurons, growth cone elongation and gene expression; thus they are crucial to the cell signalling process. They are the targets of numerous therapeutic agents, arthropod and mullusc toxins and environmentally relevant toxic metals. These channels may also be target sites in certain diseases. Lambert-Eaton Myasthenic Syndrome (LEMS) is one such disorder. It is an autoimmune disorder characterized by decreased evoked quantial release of acetylcholine (Ach) and disruption of the presynaptic active zones, the sites at which Ach is thought to be released. LEMS antibodies are thought to be directed specifically at the Ca2+ channels located at or near the active zone. During the last funding period we showed that LEMS auntoantibodies specifically reduce the function of nerve terminal Ca2+ channels and moreover shift the pattern of CA2+ channel phenotype to one expressing marked sensitivity to dihydropyridine- (DHP) type drugs. The goals then of the present applicaiton are to identify and characterize definitively the Ca2+ channel subtypes involved in the disease process, and to determine the basis for the shift in Ca2+ channel subtype during this disorder. The specific hypothesis to be tested is that exposure of nerve terminals isolated from rat brain to serum or IgG obtained from patients iwth LEMS causes destruction of P/Q subtypes of Ca2+ channels which normally control Ach release at themotor nerve ending and subsequently cause unmasking or expression of L-type DHP-sensitive channels in the axon terminal. Molecular cloning techniques, northern blot analyses of steady-state mRNa expression from sinal motor neuros and electrophysiological recordings of nerve terminal Ca2+ currents in mice which LEMS is induced by passive transfer will be studied to examine the time course and onset of the induction of DHP sensitivity as well as determine whether increased DHP-sensitivity in LEMs occurs due to enhanced expression of new L-type channels. To examine the specific types of Ca2+ channels affected by LEMS in isolation, we propose to express cloned P- and Q-type Ca2+ channels in the Xenopus oocyte expression system. We have currently isolated segments of the alpha2sigma and beta3 subunit of Ca2+ channels from human fetal spinal cord using human fetal spinal cord RNA and isolate full length copies of these genes, sequence them and engineer them with appropriate sequences for expression studies in Xenupus oocytes for 2 microelectrode voltage clamp recordings. Results of this proposal should provide information regarding the types of Ca2+ channels affected in LEMS. It will also hopefully provide structural information regarding the types of Ca2+ channel involved in neurotransmitter release- specifically from human spinal motor neurons. Results should also provide information on how neurons respond to chronic impairment of Ca2+ channel function. This may lead to strategies for treatment of disorders of Ca2+ channel function such as those seen with certain environmental toxicants, or in LEMS.
| Peng, Shuang-Qing; Hajela, Ravindra K; Atchison, William D (2005) Fluid flow-induced increase in inward Ba2+ current expressed in HEK293 cells transiently transfected with human neuronal L-type Ca2+ channels. Brain Res 1045:116-23 |
| Hajela, Ravindra K; Peng, Shuang-Qing; Atchison, William D (2003) Comparative effects of methylmercury and Hg(2+) on human neuronal N- and R-type high-voltage activated calcium channels transiently expressed in human embryonic kidney 293 cells. J Pharmacol Exp Ther 306:1129-36 |
| Flink, Michael T; Atchison, William D (2003) Ca2+ channels as targets of neurological disease: Lambert-Eaton Syndrome and other Ca2+ channelopathies. J Bioenerg Biomembr 35:697-718 |
