A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype

Abstract

TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca(2+)-permeable channel with a unique form of regulation by extracytosolic (luminal) H(+) (H(+)(e-cyto)). Regulation by H(+)(e-cyto) is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H(+)(e-cyto) to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H(+)(e-cyto), and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H(+)(e-cyto) are altered by both the alpha-helix-destabilizing A419G and the alpha-helix-favouring A419M and A419K. These findings suggest that regulation of TRPML3 by H(+)(e-cyto) is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication.

Figure 1

Channel properties of TRPML3. (A) Whole-cell current was measured in HEK cells transfected with GFP–TRPML3. The major cation in the pipette solution is 150 mM K⁺ (pH 7.3) and Ca²⁺ is buffered close to 0 with 10 mM BAPTA. The major cation in the bath solution is either140 mM Na⁺ (dark bars) or 0 Na⁺, 150 mM NMDG⁺ (light grey bars) (pH 7.4). TRPML3 was activated by exposing the extracytosolic face to a bath solution containing 0 mM Na⁺, pH 7.4. The cells were sequentially perfused with solutions containing 140 or 0 mM Na⁺, as indicated by the dark and light grey bars. The current was measured by the RAMPs protocol at −100 (closed circles) and +100 mV (open circles). (B) I/V relationships of the TRPML3 current recorded at the times shown in the large filled circles in (A). (C) TRPML3 current was measured by holding the membrane potential at −100 mV. The pipette solution is identical to that in (A) and the major ion in the bath solution is either 140 mM Na⁺ or 150 mM K⁺, as indicated above the dark bars. The dark dashed line here and in all experiments indicates the 0 current level. (D) Inward Ca²⁺ current by TRPML3 was measured with a pipette solution containing 150 mM K⁺ and 10 mM BAPTA. After measurement of the maximal Na⁺ current, the cells were exposed to 0 Na⁺ solution containing 1, 10 or 45 mM Ca²⁺. The Ca²⁺-containing bath solutions were prepared by isosmotic replacement of NMDG-Cl with CaCl₂. (E) The I/V relationships are of the TRPML3 current recorded with 140 mM Na⁺ (dark trace) or 10 mM Ca²⁺ (light grey trace) at the times indicated by the large circles in (D). (F) The reversal potential with the indicated cations in the bath solutions. K⁺, Na⁺ and Cs⁺ were at 150 mM. The results are the mean±s.e.m. of 3–5 experiments. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 2

Regulation of TRPML3 activity by extracytosolic pH (H⁺_e-cyto). (A) Na⁺ current was measured as in Figure 1A, except that at the period marked by the grey bar the cells were incubated in Na⁺-free bath solution of pH 6.0. (B) The same conditions as in (A), except that the pH of the pipette solution (pH_i) was set at 5.0. (C) H⁺_e-cyto-dependent inhibition of TRPML3-mediated Na⁺ current. The number of experiments at different H⁺_e-cyto is given next to the symbols. (D) TRPML3-mediated Ca²⁺ current was measured at 10 mM Ca²⁺_o as in Figure 1D, except that the cells were incubated in Na⁺-free bath solution of pH 5.0 (grey line) before measurement of Ca²⁺ current at pH 7.4. (E) The same conditions as in (D) were used to measure the Ca²⁺ current, except that the pH of the Ca²⁺-containing solution was 6.0. (F) H⁺_e-cyto-dependent inhibition of TRPML3-mediated Ca²⁺ current. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 3

Inhibition of TRPML3 by H⁺_e-cyto is mediated by histidine 283. (A) The position of all extracytosolic histidines in TRPML3. (B) Analysis of total and surface expression of the indicated individual, double and triple histidine mutants. (C) Na⁺ current in cells expressing TRPML3 (□) or TRPML3(H283A) (•) was measured as in Figure 2A. Note that TRPML3(H283A) is spontaneously active, does not inactivate and is not inhibited by H⁺_e-cyto. (D) The H283A mutation does not change the I/V relationship of TRPML3. (E) Effect of the H283A and A419P mutations on cell death in the presence and absence of Ca²⁺_o. Cells transfected with TRPML3, TRPML3(H283A) and TRPML3(A419P) were cultured for 24 h in DMEM media containing 1.8 mM CaCl₂ or the same media supplemented with 2.5 mM EGTA (marked 0 Ca²⁺). All cells (floating and attached) were collected to prepare cell extracts and the extracts were probed for expression of the channels. Note that the H283A and A419P mutations similarly reduced the level of TRPML3 as a result of cell death and cell death could be reduced by removal of Ca²⁺_o. (F) Ca²⁺ influx was measured in HEK cells transfected with eGFP (○), TRPML3 (▵), TRPML3(H283A) (•) and TRPML3(A419P) (▴). The cells were incubated in Ca²⁺-free medium and plasma membrane Ca²⁺ permeability was measured by increasing Ca²⁺_o to 1 mM. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 4

Effect of H252, H273 and H283R on regulation of TRPML3 by H⁺_e-cyto. (A) The H283R mutation markedly increases the rate of TRPML3 inactivation and TRPML3(H283R) is inhibited by H⁺_e-cyto. The dark dashed line in this and the other panels indicates the 0 current level. The grey line marks the period of incubation with 0 Na⁺, pH 6. (B) Na⁺ current was measured in cells expressing wild-type TRPML3 (black trace), TRPML3(H252A) (dashed trace) or TRPML3(H273A) (grey trace). Current measurement was initiated by clamping the membrane potential to −100 mV. Preincubation of wild-type, H252A and H273A TRPML3 in Na⁺-free solution resulted in full activation of the current. However, the TRPML3 current inactivated along a bi-exponential time course, with fast inactivation that was completed in 10–20 s and a slow inactivation that took a longer time (minutes). The current of the H252A and H273A mutants showed only fast inactivation. (C) The spontaneously active TRPML3(H283A) current was measured in cells continuously bathed in solution containing 140 mM Na⁺. Current is observed when switching the holding membrane potential from 0 to −100 mV. (D) TRPML3(H252A) (dashed trace) and TRPML3(H273A) (grey trace) activity is inhibited by H⁺_e-cyto. (E) Dependence of the inhibition of the TRPML3(H252A) and TRPML3(H273A) current on H⁺_e-cyto was measured at a holding potential of −100 mV. The cells were exposed to Na⁺-free medium, pH 7.4, to determine the control current and then to Na⁺-free medium of the indicated pH_o. After exposure to different Na⁺-free media of the indicated pH_o, the current was measured in media containing 140 mM Na⁺, pH 7.4. (F) Properties of the double mutants TRPML3(H252,283A) (grey trace) and TRPML3(H273,283A) (dark trace). The spontaneous activity was measured by clamping the membrane potential to −100 mV. The cells were then incubated in Na⁺-free solution, pH 7.4, and the current was measured by clamping the membrane potential to −100 mV in Na⁺-containing solution, pH 7.4. Note that the H283A mutation markedly reduces and slows down channel inactivation of H252A and H273A. A full-colour version of this figure is available at The EMBO Journal Online.

Figure 5

Effect of the varitint-waddler phenotype-causing mutation A419P on regulation of TRPML3 by H⁺_e-cyto and on cation selectivity. In all experiments, open symbols are the current at +100 mV and closed symbols are the current at −100 mV. (A) Na⁺ current of TRPML3(A419P) and its lack of inhibition by H⁺_e-cyto. (B) Na⁺ current of the double mutant TRPML3(A419P/H283R) and its lack of inhibition by H⁺_e-cyto. (C) Mean±s.e.m. of six experiments on the effect of pH_o 5.0 of the current by TRPML3, TRPML3(H283A) and TRPML3(A419P). (D) Current by wild-type TRPML3 measured at 140 mM Na⁺ and 10 mM Ca²⁺ and the I/V relationships at peak current. (E) Current of TRPML3(A419P) measured at 140 mM Na⁺ and 10 mM Ca²⁺ and the I/V relationships at peak current. (F) Mean±s.e.m of the Ca²⁺/Na⁺ selectivity of wild-type TRPML3 (n=7) and TRPML3(A419P) (n=4). A full-colour version of this figure is available at The EMBO Journal Online.

Figure 6

Mutants that destabilize and favour α-helices prevent regulation of TRPML3 by H⁺_e-cyto. In all experiments, open symbols are the current at +100 mV and closed symbols are the current at −100 mV. (A) Na⁺ current of the α-helix-destabilizing mutant A419G and its lack of inhibition by H⁺_e-cyto. (B) The spontaneous current of TRPML3(A419M), the increase in current by repeated incubation in Na⁺-free medium and the lack of regulation of TRPML3(A419M) by H⁺_e-cyto. (C) Development of the TRPML3(A419K) current by continuous application of −100 to +100 mV RAMPs in the presence of Na⁺_o and the lack of regulation of TRPML3(A419M) by H⁺_e-cyto. The results in (B) and (C) are representative of four similar experiments. A full-colour version of this figure is available at The EMBO Journal Online.