Chemical synapses without synaptic vesicles: Purinergic neurotransmission through a CALHM1 channel-mitochondrial signaling complex

Abstract

Conventional chemical synapses in the nervous system involve a presynaptic accumulation of neurotransmitter-containing vesicles, which fuse with the plasma membrane to release neurotransmitters that activate postsynaptic receptors. In taste buds, type II receptor cells do not have conventional synaptic features but nonetheless show regulated release of their afferent neurotransmitter, ATP, through a large-pore, voltage-gated channel, CALHM1. Immunohistochemistry revealed that CALHM1 was localized to points of contact between the receptor cells and sensory nerve fibers. Ultrastructural and super-resolution light microscopy showed that the CALHM1 channels were consistently associated with distinctive, large (1- to 2-μm) mitochondria spaced 20 to 40 nm from the presynaptic membrane. Pharmacological disruption of the mitochondrial respiratory chain limited the ability of taste cells to release ATP, suggesting that the immediate source of released ATP was the mitochondrion rather than a cytoplasmic pool of ATP. These large mitochondria may serve as both a reservoir of releasable ATP and the site of synthesis. The juxtaposition of the large mitochondria to areas of membrane displaying CALHM1 also defines a restricted compartment that limits the influx of Ca upon opening of the nonselective CALHM1 channels. These findings reveal a distinctive organelle signature and functional organization for regulated, focal release of purinergic signals in the absence of synaptic vesicles.

A subset of taste receptor cells express biocytin‐permeable channels activated by reducing extracellular Ca2+ concentration

“…Biocytin uptake and Cs + ‐insensitive currents were inhibited by the relatively nonselective CALHM1 blockers Gd 3+ and Zn 2+ but were not affected by another nonselective CALHM1 blocker, ruthenium red, or by pannexin 1 and Cx43 blockers. Biocytin uptake was observed after soaking the basolateral membranes of the TBCs, which is consistent with previous studies on CALHM1 channel expression (Kashio et al, ; Romanov et al, ). We confirmed the expression of CALHM1 and CALHM3 channel genes in fungiform taste buds using RT‐PCR.…”

“…CALHM channels and several types of Cx hemichannels are activated by the reduction of [Ca 2+ ] out and membrane depolarization. Recent studies have indicated that CALHM1 and CALHM1/3 complex channels are localized on the basolateral membrane in TBCs (Kashio et al, ; Romanov et al, ). In TBCs that express [Ca 2+ ] out ‐dependent channels on the basolateral membrane, extracellular biocytin is moved into the cells via the channels activated by reducing [Ca 2+ ] out .…”

“…Recently, it was reported that influx of Ca 2+ following CALHM1 activation in type II cells is difficult to detect using Ca 2+ sensitive dye because of the tight apposition of mitochondria and CALHM1 (Romanov et al, ). However, we showed that biocytin moved into the cytosol from the extracellular solution.…”

“…In this study, we showed that type II cells did not take up propidium iodide (668, +2) despite high‐K stimulation. In addition, Ca 2+ ions through the CALHM1 channels are difficult to detect with Ca 2+ sensitive dye as mentioned above (Romanov et al, ). Hence, because molar mass of propidium iodide is larger than Ca 2+ ions, it may be difficult for propidium iodide to pass through the tight apposition of mitochondria and CALHM channels by affecting both positive charge and large molar mass.…”

“…Taste substances initiate signal transduction from type II cells, which generate action potentials and then release transmitters to neighbouring TBCs or taste nerve fibres (Behe, DeSimone, Avenet, & Lindemann, ; Furue & Yoshii, ). Numerous studies have demonstrated that the predominant neurotransmitter for taste receptor cascades is ATP (Finger et al, ; Murata et al, ), which is released via connexins (Cxs) (Romanov et al, ; Romanov, Rogachevskaja, Khokhlov, & Kolesnikov, ), pannexin 1 (Dando & Roper, ; Huang et al, ), and/or calcium homeostasis modulator 1 and 3 (CALHM1 and CALHM 3) channels (Kashio, Wei‐Qi, Ohsaki, Kido, & Taruno, ; Ma et al, ; Romanov et al, ; Taruno et al, ). Among these, the CALHM channels and several types of Cx hemichannels including Cx26 and Cx43 are activated by reducing the extracellular Ca 2+ ‐concentration ([Ca 2+ ] out ) (Contreras, Saez, Bukauskas, & Bennett, ; Lopez, Gonzalez, Liu, Harris, & Contreras, ; Ma et al, ; Siebert et al, ).…”