Two isoforms of Trpm8 in rat vas deferens

Abstract

Trpm8 is a nonselective Ca 2+ -permeable ion channel activated by temperatures below 28 °C and cooling chemical compounds such as menthol and icilin. Trpm8 is expressed in sensory neurons where it functions for temperature detection. However, Trpm8 is also expressed in various internal organs where temperature is stably higher than 36 °C, that is much higher than cool temperature needed for Trpm8 activation. This opens possible makes roles for Trpm8, such as nociception. Trpm8 mRNA is expressed in vas deferens (VD), a smooth muscle organ of male reproductive system. However, no Trpm8-mediated currents were previously registered in the myocytes. VD is located outside of testes, and it consists of smooth muscle tube covered by the epithelium. It actively contracts and transfers sperm from testes to ejaculatory ducts prior to ejaculation. Temperature in VD is stably high, thus, Trpm8 role there could be outside of cold detection. The objective of this study was to analyze Trpm8 mRNA and protein expression in rat VD, as well as splice variant analysis. Trpm8 mRNA expression in VD was confirmed with RT-PCR, and Trpm8 protein was detected by the Western-blot analysis. Additionally, we found that both mRNA and protein of shorter non-classical isoform, as well as canonical isoform of Trpm8 in VD. We isolated smooth muscle cells from VD and performed a multi-cell PCR. This technique makes possible non-myocytic mRNA detection that could be isolated from e.g. sensory neurons termini where Trpm8 is expressed at much higher levels. Interestingly, in the isolated smooth muscle cells, no canonical Trpm8 transcript was found, though the non-classical isoform was present. We propose the shorter isoform could be formed as a result of alternative splicing. This would account for a difference in Trpm8 function in VD, i.e. no Trpm8-mediated currents registered in the myocytes. A shorter isoform could have a truncated N-terminal domain, that is consistent with known human Trpm8 isoforms sM8-6 and sM8-18.