Properties of the TRPML3 channel pore and its stable expansion by the Varitint-Waddler-causing mutation

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.

FIGURE 1.

Dynamic behavior of the TRPML3 pore. a, whole cell current was measured in HEK cells transfected with WT TRPML3. The major cation in the pipette solution was 150 mm Cs⁺, and Ca²⁺ was buffered close to 0 with 10 mm BAPTA. The major cation in the bath solution was 150 mm Na⁺ (black bars), 150 mm NMDG⁺ (light gray bars), or 100 mm Ca²⁺ (black bars). TRPML3 was activated by exposing the cells to Na⁺-free bath solution. After measurement of the maximal Na⁺ current, the cells were exposed to 100 mm Ca²⁺. Shown is the current measured at −100 (lower circles) and +100 mV (upper circles). Dashed lines here and in all other figures indicate the 0 current levels. b and c, I/V relationships of the TRPML3 current recorded at the times shown in the large black circles in a. d, changes in E_rev during the first 1 min of WT TRPML3 in 150 Na⁺ (squares) and 100 Ca²⁺ (circles). The results are the mean ± S.E. of seven experiments. e, current clamp protocol was used to record the membrane potentials.

FIGURE 2.

Effect of the Ca²⁺ buffer and current density on E_rev. Cells expressing TRPML3 were dialyzed with pipette solutions containing either 10 μm Ca²⁺ (2 mm EGTA + 2 mm CaCl₂) (a) or 10 mm BAPTA (b). The current was recorded every second by the RAMPs protocol described under “Materials and Methods.” The figure shows example traces with two different current densities. The average time constants obtained from single exponential fits are given as mean ± S.E. of the indicated number of experiments.

FIGURE 3.

Modulation of TRPML3 activity by Ca²⁺. a, Ca²⁺ current of WT TRPML3 was measured as in Fig. 1a, except that the holding potential (HP) was +80 mV. b, E_rev changes during the first 1 min of current recording for WT TRPML3 in 100 Ca²⁺ solutions at holding potentials of +80 mV (filled circles) and 0 mV (open circles). The latter is reproduced from Fig. 1d to facilitate comparison. The results at +80 mV are the mean ± S.E. of three experiments. c, Na⁺ and Ca²⁺ currents of WT TRPML3 were measured as in Fig. 1, except that at the periods marked by gray bars the cells were incubated in 10 mm Ca²⁺-containing Na⁺-free bath solution. All Na⁺-free solutions contained 5 mm EGTA. Note that after conducting Ca²⁺, both Na⁺ and Ca²⁺ currents are strongly inhibited. d, mean ± S.E. (error bars) of the E_rev recorded at the times shown in the large filled circles in c in 10 mm Ca²⁺ during the first and second exposure to Ca²⁺ and the recovery after relief of the inhibition by conduction of Na⁺ by the channel (n = 4). The first columns are the initial E_rev, and the striped columns are the E_rev after 30-s exposure to 10 mm Ca²⁺.

FIGURE 4.

Effect of the varitint-waddler phenotype causing A419P mutation on TRPML3 pore behavior. a and e, whole cell currents of the A419P and H283A mutants were measured as in Fig. 1. b and c, f and g, I/V relationships for each mutant recorded at the times shown by the large filled circles in a and e. d and h, changes in E_rev for the first 1 min of current recording for each mutant. Open symbols are the E_rev for WT TRPML3. Error bars, S.E.

FIGURE 5.

Effect of fifth transmembrane domain mutations on the TRPML3 pore behavior. a, whole cell current of the A419G mutant was measured as in Fig. 1. b and c, I/V relationships for TRPML3(A419G) recorded at the times shown by the large filled circles in a. d–h, E_rev changes for the first 1 min for each mutant. Open symbols are the E_rev changes for TRPML3(A419P). Error bars, S.E.

FIGURE 6.

Permeation profile of monovalent cations for WT and mutants TRPML3. a–f, monovalent currents and I/V relationships were measured in extracellular solutions containing a 150 mm concentration of the indicated monovalent cation. Note the different permeability sequence for the TRPML3(A419P). g, summary of the relative permeability for WT and mutants TRPML3. The results are the mean ± S.E. (error bars) of 8–10 experiments.

FIGURE 7.

Measurement of pore diameter. a–c, HEK cells expressing the indicated TRPML3 constructs were exposed to extracellular solutions containing either 150 mm Na⁺ or the indicated organic monovalent cations. d, permeability of the organic cations relative to Na⁺ is plotted against the size of each cation (n = 4–5).