CALHM1/CALHM3 channel is intrinsically sorted to the basolateral membrane of epithelial cells including taste cells

Makiko Kashio,Akiyuki Taruno,Mizuho A Kido,Gao Wei-Qi,Yasuyoshi Ohsaki

doi:10.1038/s41598-019-39593-5

Abstract

The CALHM1/CALHM3 channel in the basolateral membrane of polarized taste cells mediates neurotransmitter release. However, mechanisms regulating its localization remain unexplored. Here, we identified CALHM1/CALHM3 in the basolateral membrane of type II taste cells in discrete puncta localized close to afferent nerve fibers. As in taste cells, CALHM1/CALHM3 was present in the basolateral membrane of model epithelia, although it was distributed throughout the membrane and did not show accumulation in puncta. We identified canonical basolateral sorting signals in CALHM1 and CALHM3: tyrosine-based and dileucine motifs. However, basolateral sorting remained intact in mutated channels lacking those signals, suggesting that non-canonical signals reside elsewhere. Our study demonstrates intrinsic basolateral sorting of CALHM channels in polarized cells, and provides mechanistic insights.

Highlights

Calcium homeostasis modulator 1 (CALHM1) and its homolog, CALHM3, hetero-hexamerize to form a non-selective fast-activating voltage-gated channel, CALHM1/CALHM3, which is permeable to large molecules including ATP1
As plasma membrane proteins cannot diffuse over the tight junction, CALHM1/CALHM3 must initially be delivered to the basolateral membrane, and subsequently accumulate at points of contact with nerve fibers
Immunofluorescence staining of tongue sections containing circumvallate papillae using an antibody targeting the carboxyl terminus (Cter) end of mouse CALHM1 (Fig. 1A,B) revealed small punctate signals within the wild-type taste buds (Fig. 1C,D)

Summary

Introduction

Calcium homeostasis modulator 1 (CALHM1) and its homolog, CALHM3, hetero-hexamerize to form a non-selective fast-activating voltage-gated channel, CALHM1/CALHM3, which is permeable to large molecules including ATP1. In response to taste stimuli applied to the apical membrane, type II TBCs generate action potentials in the basolateral membrane, which lead to the release of ATP as the neurotransmitter towards gustatory nerves expressing the ATP-gated ion channel P2X2/3R15. Another study reported CALHM1 localization in the basolateral membrane of type II TBCs at points of contact with P2X2R-expressing nerve fibers for the focal release of purinergic signals[16]. We generated an antibody against a short peptide sequence corresponding to the carboxyl terminal end of mouse CALHM1, and data from immunohistological analyses using it supported punctate localization near nerve fibers in the basolateral membrane of type II TBCs16. Using an epithelial model of MDCKII cells, we explored the mechanisms of the polarized sorting of CALHM1/CALHM3 to further understanding of the structural basis behind the regulation of CALHM channel localization in polarized cells

Methods

Results

Conclusion