Corticolimbic Expression of TRPC4 and TRPC5 Channels in the Rodent Brain

Melissa A Fowler,Kyriaki Sidiropoulou,Donald C Cooper,Christopher W Phillips,Emin D Ozkan,Brian Mccabe

doi:10.1371/journal.pone.0000573

Abstract

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca2+ and Gq/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6–9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2–6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca2+and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.

Highlights

The dynamic homeostatic mechanisms that neurons use to regulate intracellular Ca2+ signaling have received much attention recently, due to the important role Ca2+ plays in cellular processes including gene expression, axon growth, synaptic plasticity and cell death
Immunoblots performed for TRPC4 and 5 protein in microdissections of HIP from post-natal day 0 (P0) and post-natal day 48 (P48) mice show that TRPC4 and 5 protein levels are higher at P48 compared to P0 (Figure 8C)
Similar results were observed in rat prefrontal cortex (PFC), ventral subiculum (vSUB), and ECx comparing postnatal day 21 (P21) and post-natal day 63 (P63) rats (Figure 9)

Summary

Introduction

The dynamic homeostatic mechanisms that neurons use to regulate intracellular Ca2+ signaling have received much attention recently, due to the important role Ca2+ plays in cellular processes including gene expression, axon growth, synaptic plasticity and cell death. The TRPC non-selective cation channels consist of seven members that are organized into four groupings based on sequence homology and functional similarities: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5 [2],[3]. These channels are mixed cation (K+, Na+ and Ca2+) channels that are activated by Gqcoupled receptors, such as group 1 mGluR and muscarinic acetylcholine receptors [4],[5],[2],[6],[7],[8]. A conformational change opens the TRPC channel bound to IP3 receptors, which bind to the C-terminal end of the TRPC channels via the calmodulin/IP3 receptor binding domain [12],[14]

Methods

Results

Conclusion