The long term goal of this project is to gain a better understanding of the molecular biology of the pathogenesis of Lambert-Eaton Myasthenic Syndrome (LEMS), a prejunctionally-directed disorder of neuromuscular transmission frequently associated with oat cell carcinoma of the lung. LEMS is associated with decreased number of packets of acetylcholine (ACh) released in response to a nerve impulse and decreased number of particles associated with the active zone of the presynaptic nerve terminal membrane, sites at which ACh is thought to be released. LEMS is thought to be due to circulating autoantibodies to some component of the presynaptic motor nerve terminal. Knowledge of the clear identity and physicochemical characteristics of the polypeptide targets of these antibodies is critical to a better understanding of the biology of this disease. Based on studies in several model systems the target is believed to be the calcium (Ca2+) channel in the membrane of the presynaptic nerve terminal responsible for permitting Ca2+ entry during nerve excitation, leading to subsequent release of ACh. The immediate goal of this project is the identification and molecular characterization of the proposed site of action of the LEMS autoantibody on the nerve terminal. A model system of isolated nerve terminals from the central nervous system (synaptosomes) of the rat will be used. The specific hypothesis under test is that acute exposure of nerve terminals to serum or IgG obtained from patients with LEMS impairs the function of voltage-dependent Ca2+ channels of isolated nerve terminals. Experiments proposed are designed with the intent of identify proteins in the nerve terminal membrane to which LEMS IgG binds.
The first aim i s to determine if immunoglobulins isolated from patients with LEMS bind to a protein(s) that is (are) associated with the voltage-gated Ca2+ channel complex in isolated nerve terminals.
The second aim i s to determine if the proteins in the nerve terminal membrane recognized by LEMS IgG are identical to those bound by known Ca2+ channel ligands, and thus identify the type of Ca2+ channel affected. The binding of LEMS IgG to western blots of synaptosomal proteins immobilized by one and two dimensional polyacrylamide gel electrophoresis will be used to determine whether the autoantibody binds to """"""""N'~, """"""""L"""""""" or """"""""P"""""""" type Ca2+ channels. Inferences regarding the Ca2+ channel subunit affected will be made based on molecular weight and isoelectric point determinations. Attempts will be made to immunoaffinity purify the target proteins for more detailed studies. The proposed research will begin to determine and characterize the site(s) on nerve terminals to which the LEMS autoantibody binds. This information will be important in determining the molecular target(s) mediating the pathogenesis of LEMS with a view towards ultimately probing the mechanisms involved in the disease process, including factors which may predispose an individual to the autoimmune response.
| Hajela, R K; Atchison, W D (1998) Isolation of Ca2+ channel alpha 1A, alpha 2, and beta subunit segments from human spinal cord RNA. Ann N Y Acad Sci 841:115-8 |
| Hajela, R K; Atchison, W D (1995) The proteins synaptotagmin and syntaxin are not general targets of Lambert-Eaton myasthenic syndrome autoantibody. J Neurochem 64:1245-51 |
