Submitted to: Aging Cell
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/17/2004
Publication Date: 10/1/2004
Citation: Joseph, J.A., Fisher, D.R., Carey, A.N., Szprengiel, A. 2004. The m3 muscarinic receptor i3 domain confers oxidative stress protection on calcium regulation in transfected cos-7 cells. Aging Cell (2004)3, pp263-271. Interpretive Summary: neurodegenerative diseases of aging such as Alzheimer disease (AD) is an increased sensitivity to very reactive molecules called free radicals. The free radicals, since many of them require oxygen, produce oxidative stress (OS) which can have a variety of negative effects on the brain and elsewhere in the body. This could eventually lead to a loss of function in neuronal cells, as has been observed in aging and diseases such as AD. Recently, we showed in cells that different subtypes of a neurotransmitter receptors called muscarinic receptors, which are important in the regulation of calcium, respond differently to agents which produce OS. Some of the muscarinic receptor subtypes when placed in cells increased the sensitivity of agents which produce OS, such as dopamine. This caused the cells to lose their ability to regulate calcium levels when they were stimulated. Interestingly, similar effects were seen when we used amyloid beta to produce OS in the cells. This peptide makes up the characteristic plaques in the brains of AD patients. A large number of studies have shown that this peptide is toxic in cells, and may have similar toxic effects in the brain. In this study we found that when we used molecular biological techniques to alter the structures of the sensitive and non-sensitive receptor subtypes and placed them into the cells, one part of the structure called the i3 loop was important. This loop controls communication in the receptor and also is one of the regulators of calcium activation in the cell. These findings indicate that there may be differences in the structures of the various muscarinic receptor subtypes which could provide some protection from or impart vulnerability to oxidative stress. Future studies will investigate the factors in the loop that could increase OS sensitivity (e.g., amino acids etc.) with a view toward identifying the possible receptor locus (i) of dietary antioxidants in their protective capabilities.
Technical Abstract: Evidence suggests that muscarinic receptors (MAChRs) are intimately involved in various aspects of both neuronal and vascular functioning, and that there is a selective oxidative stress sensitivity (OSS) among MAChR subtypes, with M1, M2, and M4 showing greater OSS [as seen by the ability of the cell to extrude or sequester Ca2+ following depolarization by 750 ÿM oxotremorine and exposure to dopamine DA(1mM/4 hrs) or Aÿ 25-35 (100 ÿM/24hrs)] than M3 or M5 subtypes in transfected COS-7 cells. We sought to determine the receptor domain in an OSsensitive (M1) and non-sensitive (M3) subtype that may be responsible for the differential vulnerability to OS between receptor subtypes. Amino acid sequencing of the receptors has shown the third cytoplasmic loop (i3 loop) to be the domain with the most variability between M1 and M3. Therefore, mutations were made by deleting segments (up to 400 base pairs) along the M1 and M3 receptors, focusing primarily in the i3 loop. Small deletions of a 128bp piece near the N-terminal region of the i3 loop in rat M1 receptors transfected into COS-7 cells showed no differences in % of the cells showing recovery following DA treatment, i.e., the mutated receptors showed no significant increase or decrease in their ability to protect against the deleterious effect of DA on % of the cells showing recovery following depolarization. Additional studies showed that deletions of the entire i3 loop increased DA sensitivity (a lower % of cells showing recovery following depolarization) in both the M1 and M3 subtypes. Chimerics of M1 where the i3 loop of the M3AChR was switched with the i3 loop of the M1AChR (M1M3i3) showed that the DA sensitivity was reduced (% cells showing increases in calcium clearance) following depolarization. In the M3 chimerics containing M1i3 (M3M1i3), the i3 loop offered no protection against DA-induced decrements in calcium buffering. Results are discussed in terms of the length of the i3 loop in mediating OS sensitivity and the possible targeting of antioxidants to specific receptor sites that impart oxidative stress sensitivity.