Extracellular Acid Block and Acid-enhanced Inactivation of the Ca2+-activated Cation Channel TRPM5 Involve Residues in the S3-S4 and S5-S6 Extracellular Domains

Dan Liu,Zheng Zhang,Emily R Liman

doi:10.1074/jbc.m414072200

Dan Liu, Zheng Zhang + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m414072200

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Abstract

TRPM5, a member of the superfamily of transient receptor potential ion channels, is essential for the detection of bitter, sweet, and amino acid tastes. In heterologous cell types it forms a nonselective cation channel that is activated by intracellular Ca(2+). TRPM5 is likely to be part of the taste transduction cascade, and regulators of TRPM5 are likely to affect taste sensation. In this report we show that TRPM5, but not the related channel TRPM4b, is potently blocked by extracellular acidification. External acidification has two effects, a fast reversible block of the current (IC(50) pH = 6.2) and a slower irreversible enhancement of current inactivation. Mutation of a single Glu residue in the S3-S4 linker and a His residue in the pore region each reduced sensitivity of TRPM5 currents to fast acid block (IC(50) pH = 5.8 for both), and the double mutant was nearly insensitive to acidic pH (IC(50) pH = 5.0). Prolonged exposure to acidic pH enhanced inactivation of TRPM5 currents, and mutant channels that were less sensitive to acid block were also less sensitive to acid-enhanced inactivation, suggesting an intimate association between the two processes. These processes are, however, distinct because the pore mutant H896N, which has normal sensitivity to acid block, shows significant recovery from acid-enhanced inactivation. These data show that extracellular acidification acts through specific residues on TRPM5 to block conduction through two distinct but related mechanisms and suggest a possible interaction between extracellular pH and activation and adaptation of bitter, sweet, and amino acid taste transduction.

Highlights

Channel TRPM5 are essential elements [5]
TRPM5 Is Blocked by Extracellular Acidification—When expressed in HEK 293 cells, mTRPM5 forms a cation-selective conductance that is activated by elevation in intracellular Ca2ϩ
Activation of mTRPM5 is detected by voltage ramps (Ϫ80 to ϩ80 mV), which elicit a characteristic outwardly rectifying current in mTRPM5-expressing cells that are dialyzed with 40 ␮M Ca2ϩ (Fig. 1) (18 –20)

Summary

Introduction

Channel TRPM5 are essential elements [5]. It is likely that upon binding of taste molecules, sweet, bitter, and amino acid taste receptors initiate a signaling cascade that activates phospholipase C ␤2, leading to the breakdown of phosphatidylinositol 4,5-bisphosphate into inositol trisphosphate and diacylglycerol and the release of Ca2ϩ from intracellular stores [6, 7]. TRPM5 and TRPM4 are activated by Ca2ϩ [17,18,19,20,21], and both channels likely underlie the Ca2ϩ-activated cation currents observed in a variety of native tissues TRPM5 channels are activated by Ca2ϩ with an EC50 of 20 ␮M and undergo a process of Ca2ϩ-dependent desensitization, which is partially reversed by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [20]. TRPM5 currents are activated by micromolar concentrations of Ca2ϩ and show pronounced outward rectification, which is due to voltage-dependent gating of the channels [18, 20]. Neither divalent nor trivalent cations block TRPM5 currents. Proton block is conserved among human and mouse TRPM5 but is not observed for human TRPM4b. We identify critical residues that differ between TRPM4b and TRPM5 that mediate this block

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