Extracellular Zinc Ion Regulates Transient Receptor Potential Melastatin 5 (TRPM5) Channel Activation through Its Interaction with a Pore Loop Domain

Kunitoshi Uchida,Makoto Tominaga

doi:10.1074/jbc.m113.470138

Abstract

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca(2+). Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. Its endogenous ligands are not well characterized. Here, we show that extracellular application of Zn(2+) inhibits TRPM5 activity. In whole-cell patch-clamp recordings, extracellular application of ZnCl2 inhibited step-pulse-induced TRPM5 currents with 500 nM free intracellular Ca(2+) in a dose-dependent manner (IC50 = 4.3 μM at -80 mV). ZnSO4 also inhibited TRPM5 activity. Extracellular application of ZnCl2 inhibited TRPM5 activation at several temperatures. Furthermore, inhibition by 30 μM ZnCl2 was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln. From these results, we conclude that extracellular Zn(2+) inhibits TRPM5 channels, and the residues in the outer pore loop of TRPM5 are critically involved in the inhibition.

Highlights

transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca2ϩ
Inhibition by 30 ␮M ZnCl2 was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln
TRPM5 activity was inhibited in a dose-dependent manner, similar at both positive and negative potentials, and the dose-dependent curves could be fit to a logistic function with IC50 values of 4.3 Ϯ 3.0 ␮M at Ϫ80 mV and 4.3 Ϯ 0.7 ␮M at ϩ160 mV (Fig. 1B)

Summary

Background

TRPM5 channel is a monovalent cation channel activated by intracellular Ca2ϩ. Results: TRPM5 was inhibited by extracellular Zn2ϩ. The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca2؉. Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. TRPM5 is thought to function as a tetramer of each subunit containing intracellular N and C termini, 6 transmembrane (TM) domains, and a pore-forming loop between TM5 and TM6 like other TRP channels [1, 2] This channel is restrictedly expressed in taste cells, the pancreas, and the brainstem, and is thought to be involved in controlling membrane potentials [2]. We determined the amino acid residues required for the inhibition

MATERIALS AND METHODS

RESULTS

DISCUSSION