IDENTIFICATION OF THE α7 NICOTINIC ACETYLCHOLINE RECEPTOR CHAPERONE PROTEIN RIC-3 IN THE MAMMALIAN BRAIN: EVIDENCE FOR ISOFORMS AND MULTIPLE FUNCTIONS

Ruan van Rensburg1, Abdel Ennaceur2, Paul Chazot1
1Durham University, Durham, United Kingdom, 2Sunderland Pharmacy School, Sunderland, United Kingdom

There is growing evidence that the α7-nACh receptor plays a significant role in human cognitive and emotional behaviours, and is currently a popular putative therapeutic target for a range of acute and chronic neuropathologies. Therefore, regulation of functional expression of α7-nAChR clearly has significant pharmacological implications. Ric-3 (resistant to inhibitors of cholinesterase) has been recently identified as an obligatory trafficking chaperone protein for the α7 nACh and 5HT3 receptors in heterologous expression studies in vitro – a link that remains to be confirmed in vivo.

As part of our strategy to address this issue, we have developed the first antibody for the rodent Ric-3 protein, which has been used to identify Ric-3 in rat and mouse brain. Based on immunoblotting (Chazot et al., 2002), our new anti-mRic-3 (208-222) polyclonal antibody, identifies a common diffuse protein species (Mr 50-55 kDa) expressed in the mouse and rat brain, together with a prominent Mr 25,000 species in the rat brain, which is expressed at low levels in the adult mouse brain. The size of the smaller protein compares well to the published mouse homolog, while the larger protein species is likely to be a rodent Ric-3 isoform (a potential homolog of the major human isoform). The anti-mRic-3 antibody was also used to define the anatomy of Ric-3 protein expression in the mammalian brain, using a standard immunohistochemistry technique (Chazot et al., 2001). Many important brain structures expressed high levels of Ric-3, including specific subfields (CA1, CA3 and Dentate gyrus) of the hippocampal formation, and outer laminae II of the cerebral cortex. This expression pattern matches well with the α7 nAChR. Notable high expression is also found in the thalamic and hypothalamic paraventricular nuclei, as well as in the perhinal cortex – structures implicated in both emotional and cognitive behaviours. Interestingly, Ric-3 was also highly expressed in the mouse choroid plexus, a non-neuronal cell type not known to contain α7 nAChRs, indicating additional roles for this protein.

Therefore, we provide the first evidence for at least two rodent Ric-3 isoforms, and the expression of Ric-3 in non-neuronal epithelial cells, indicating further a distinct role for Ric-3 independent of α7 nAChRs.

PL Chazot et al. (2001) NeuroReport 12, 259-262.
PL Chazot et al. (2002) J. Neurochem. 83, 1235-1238.

This study is funded by the BBSRC (UK), Durham School of Health, the ORS and NRF (South Africa).