Properties of the TRPML3 Channel Pore and Its Stable Expansion by the Varitint-Waddler-causing Mutation

Hyun Jin Kim,Soichiro Yamaguchi,Qin Li,Insuk So,Shmuel Muallem

doi:10.1074/jbc.m109.078204

Abstract

TRPML3 is a H(+)-regulated Ca(2+) channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca(2+) to change its conductance and permeability, which appears to be mediated by trapping Ca(2+) within the pore. The pore properties can be restored by strong depolarization or by conducting Na(+) through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca(2+). This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype.

Highlights

TRPML3 functions as an inwardly rectifying cation channel [6], with high Ca2ϩ/Kϩ selectivity [7,8,9,10] and shuttles between the plasma membrane and intracellular organelles to regulate membrane trafficking [11, 12] and autophagy [11]
We reported a unique form of regulation of TRPML3 by extracytosolic Naϩ and Hϩ (Hϩe-cyto), which is mediated by a string of three histidines (His252, His273, and His283) in the large extracytosolic loop between TMD1 and TMD2 [7]
Monovalent ion solutions conand the A419G in the fifth transmembrane domain (TMD), do not affect pore behavior. tained 150 mM monovalent ions instead of NMDGϩ in the These findings describe a unique behavior of the TRPML3 pore Naϩ-free solution, adjusted to pH 7.4 with NMDG-OH

Summary

Introduction

TRPML3 functions as an inwardly rectifying cation channel [6], with high Ca2ϩ/Kϩ selectivity [7,8,9,10] and shuttles between the plasma membrane and intracellular organelles to regulate membrane trafficking [11, 12] and autophagy [11]. Because proline introduces a kink or a break in ␣-helical transmembrane spans, and the A419G mutation that may disrupt ␣-helical structures results in a similar GOF in TRPML3 as the A419P [6, 9], it was postulated that the GOF by TRPML3(A419P) is caused by destabilization of the fifth TMD helix of TRPML3 [9, 13] It is still unclear how the proline-induced destabilization of the fifth TMD affects the behavior of the TRPML3 pore to lead to the GOF. The pore of the wild-type (WT) channel shows high Ca2ϩ/Kϩ and Ca2ϩ/Csϩ selectivity with similar Naϩ and Ca2ϩ conductance at isotonic ion concentrations [7]. Ligand stimulation dilates the TRPA1 pore [19, 20] and increases its Ca2ϩ/Naϩ selectivity ratio [19]

Methods

Results

Conclusion