Production of antisera selective for m1 muscarinic receptors using fusion proteins: distribution of m1 receptors in rat brain

Abstract

A fragment of the cDNA encoding the third intracellular loop of the rat m1 muscarinic receptor was cloned, and the DNA was expressed in Escherichia coli as a fusion protein. The fusion protein was purified and utilized as an antigen to raise a polyclonal antiserum in rabbits. Chinese hamster ovary cells stably transfected with the cDNA encoding each of the five known subtypes of muscarinic receptor were used as tissue sources to test the antiserum. The antiserum was found to quantitatively immunoprecipitate m1 muscarinic receptors, while not precipitating m2, m3, m4, or m5 receptors. This selective antiserum was utilized to quantify the density of m1 muscarinic receptors in seven selected areas of the rat brain. Thus, cortex was found to contain approximately 0.8 pmol/mg of membrane protein, which represents 34% of the total density of muscarinic receptors. Similarly, hippocampus (1 pmol/mg; 47%), striatum (0.8 pmol/mg; 29%), and olfactory tubercule (0.9 pmol/mg; 35%) are rich in m1 receptors. In contrast, thalamus/hypothalamus contained only 0.15 pmol/mg, representing approximately 16% of the total density of muscarinic receptors, whereas pons/medulla (0.03 pmol/mg; 5%) and cerebellum (less than 0.01 pmol/mg; 2%) had very low levels of expression of m1 receptors. The development of a selective antiserum has provided a means for the quantification of a specific subtype of muscarinic receptor in tissues, such as the brain, that express multiple subtypes. This methodology will be applicable not only to the other subtypes of muscarinic receptor but also to the subtypes of several other neurotransmitter receptors that lack selective drugs with which to study them.