Subtype selective toxins as tools for investigating M1 and M4 muscarinic acetylcholine receptors

Ezra Mulugeta¹, Irwin Chandranath², Abdu Adem^1,2, Evert Karlsson¹

1 Department of Clinical Neuroscience, Experimental Geriatrics, Karolinska Institute, 141 86 Huddinge, Sweden; 2 Department of Pharmacology, Faculty of Medicine and Health Sciences, P.O. Box 17 666, Al Ain, United Arab Emirates.

There are 5 muscarinic acetylcholine receptors M1 to M5 which regulate many different processes, for instance the function of smooth muscles and glands, rate and force of the heart beat, release of neurotransmitters, gene expression, migration of epithelial cells (e.g. at wound healing), pain transmission and cognitive functions such as learning and memory.

To study the role of the subtypes specific ligands are very useful. The most specific muscarinic ligands are toxins from mamba venoms against M1, M2 and M4 and derivatives of the alkaloid brucine against M3 receptors.

Two experiments will be discussed. The ligands were two mamba toxins,¹²⁵I- MT7 (selective for M1) or ¹²⁵I-MT3 (for M4).

Toxin binding was determined by quantitative autoradiography.

1) Adrenalectomyof rats ( removal of adrenal glands) is known to produce memory impairment. 10 µm sections were cut from brains and incubated with radioactive toxins. Controls were brains from Sham operated rats (Sham = operated without removal of adrenal glands).

M1 receptors up to 150 days after operation remained constant in entorhinal cortex and in the various regions of hippocampus, whereas M4 receptors decreased significantly. This suggests a role for M4 receptors in memory.

2) Animal model of pain; rats made arthritic by intradermal injection of  heat-killed Mycobacterium butyricum. Sections from lumbar regions of the spinal cord were cut for autoradiography.

M1 receptors were not detected in spinal cord. M4 receptors decreased by more than 90% as compared to controls. Muscarinic receptors are coupled to G-proteins, such receptors cycle between an extra and intracellular location. Agonist stimulation decreases extracellular receptors. Pain evidently produced a long-term release of acetylcholine which decreased M4 receptors on the cell membrane. M4 receptors are involved in pain transmission.