Mouse taste cells with G protein-coupled taste receptors lack voltage-gated calcium channels and SNAP-25

Abstract

Background: Taste receptor cells are responsible for transducing chemical stimuli from the environment and relaying information to the nervous system. Bitter, sweet and umami stimuli utilize G-protein coupled receptors which activate the phospholipase C (PLC) signaling pathway in Type II taste cells. However, it is not known how these cells communicate with the nervous system. Previous studies have shown that the subset of taste cells that expresses the T2R bitter receptors lack voltage-gated Ca2+ channels, which are normally required for synaptic transmission at conventional synapses. Here we use two lines of transgenic mice expressing green fluorescent protein (GFP) from two taste-specific promoters to examine Ca2+ signaling in subsets of Type II cells: T1R3-GFP mice were used to identify sweet- and umami-sensitive taste cells, while TRPM5-GFP mice were used to identify all cells that utilize the PLC signaling pathway for transduction. Voltage-gated Ca2+ currents were assessed with Ca2+ imaging and whole cell recording, while immunocytochemistry was used to detect expression of SNAP-25, a presynaptic SNARE protein that is associated with conventional synapses in taste cells.

Results: Depolarization with high K+ resulted in an increase in intracellular Ca2+ in a small subset of non-GFP labeled cells of both transgenic mouse lines. In contrast, no depolarization-evoked Ca2+ responses were observed in GFP-expressing taste cells of either genotype, but GFP-labeled cells responded to the PLC activator m-3M3FBS, suggesting that these cells were viable. Whole cell recording indicated that the GFP-labeled cells of both genotypes had small voltage-dependent Na+ and K+ currents, but no evidence of Ca2+ currents. A subset of non-GFP labeled taste cells exhibited large voltage-dependent Na+ and K+ currents and a high threshold voltage-gated Ca2+ current. Immunocytochemistry indicated that SNAP-25 was expressed in a separate population of taste cells from those expressing T1R3 or TRPM5. These data indicate that G protein-coupled taste receptors and conventional synaptic signaling mechanisms are expressed in separate populations of taste cells.

Conclusion: The taste receptor cells responsible for the transduction of bitter, sweet, and umami stimuli are unlikely to communicate with nerve fibers by using conventional chemical synapses.

Figure 1

Laser scanning confocal micrographs (LCSMs) of mouse circumvallate taste buds show that GFP is an accurate reporter of protein. Panels A-C show sections from transgenic mice expressing GFP under the control of the T1R3 promoter. Green denotes T1R3 GFP expression (A, C) and red shows T1R3-like-immunoreactivity (LIR) (B, C). Panels D-F show sections taken from mice expressing GFP under control of the TRPM5 promoter. Green denotes TRPM5 GFP expression (D, F) and red shows TRPM5-LIR (E, F). Scale bar is 20 μm.

Figure 2

A depolarizing stimulus of high K⁺ solution does not increase [Ca²⁺]_i in bitter, sweet, and umami sensitive cells. Panel A shows an isolated non-GFP cell from a T1R3-GFP expressing animal. Some non-GFP cells do present an increase in [Ca²⁺]_i when challenged with high K⁺ solution. Cells that have [Ca²⁺]_i responses to high K⁺ do not respond to the PLC agonist m-3M3FBS, 10 μM. T1R3-GFP expressing cells in Panel B do not show changes in [Ca²⁺]_i to bath applied high K⁺ but do show reversible increases in [Ca²⁺]_i to bath applied PLC agonist m-3M3FBS (10 μM). TRPM5-GFP expressing cells in Panel C do not change [Ca²⁺]_i to high K⁺ but show reversible increases in [Ca²⁺]_i when 10 μM m-3M3FBS is present.

Figure 3

Whole cell voltage clamp recordings from a T1R3-GFP expressing taste cell (left panel) and a non-GFP-expressing taste cell (right panel). Holding potential was -80 mV, and the membrane was stepped from -80 to +60 mV to elicit voltage-gated currents. Top panels show recordings in Tyrode's, while middle panels show recordings in Ba²⁺ Tyrode's to reveal Ba²⁺ currents through voltage gated Ca²⁺ channels. Bottom panels illustrate the current/voltage relationship.

Figure 4

LCSMs from mouse circumvallate papillae showing T1R3 promoter driven GFP expression and SNAP-25-LIR. Green denotes GFP expression (A, C) and red SNAP-25-LIR (B, C). The GFP+ cells appear to be a separate population than those with SNAP-25-LIR. Scale bar is 20 μm.

Figure 5

Z-series stack of five LCSMs 0.5 μm apart from mouse circumvallate papillae showing TRPM5 promoter driven GFP expression and SNAP-25-LIR. Green denotes GFP expression (A, C) and red SNAP-25-LIR (B, C). The GFP+ cells appear to be a separate population than those with SNAP-25-LIR. Scale bar is 20 μm.