Agonist-induced changes in Ca(2+) permeation through the nociceptor cation channel TRPA1

Abstract

The Ca(2+)-permeable cation channel TRPA1 acts as an ionotropic receptor for various pungent compounds and as a noxious cold sensor in sensory neurons. It is unclear what proportion of the TRPA1-mediated current is carried by Ca(2+) ions and how the permeation pathway changes during stimulation. Here, based on the relative permeability of the nonstimulated channel to cations of different size, we estimated a pore diameter of approximately 11 A. Combined patch-clamp and Fura-2 fluorescence recordings revealed that with 2 mM extracellular Ca(2+), and at a membrane potential of -80 mV, approximately 17% of the inward TRPA1 current is carried by Ca(2+). Stimulation with mustard oil evoked an apparent dilatation of the pore of 3 A and an increase in divalent cation selectivity and fractional Ca(2+) current. Mutations in the putative pore that reduced the divalent permeability and fractional Ca(2+) current also prevented mustard-oil-induced increases in Ca(2+) permeation. It is interesting that fractional Ca(2+) currents for wild-type and mutant TRPA1 were consistently higher than values predicted based on biionic reversal potentials using the Goldman-Hodgkin-Katz equation, suggesting that binding of Ca(2+) in the pore hinders monovalent cation permeation. We conclude that the pore of TRPA1 is dynamic and supports a surprisingly large Ca(2+) influx.

Figure 1

Monovalent cation permeability of TRPA1. (A) Current-voltage (IV) relations for anorganic monovalent cation currents through nonstimulated TRPA1 channels. The internal solution contained 150 mM Na⁺ and the external solutions contained 150 mM of the indicated cation. (B) P_X/P_Na values for the different anorganic monovalent cations obtained from the reversal potential of experiments in A. (C and D) IV relations for external solutions containing 150 mM of Na⁺ or the indicated organic cations obtained before (C) and during (D) stimulation with 20 μM MO. (E) Plot of the P_X/P_Na values for the different (an)organic cations versus their minimum diameters. For the organic cations, values were determined both before (black) and during (red) stimulation with 20 μM MO. Solid lines represent best fits to the data for the four largest cations, using Eq. 3, which yielded pore diameters of 11.0 and 13.8 Å before and during MO stimulation, respectively. Each data point represents the mean ± SE from at least five cells.

Figure 2

TRPA1-dependent uptake of FM1-43. (A) Representative brightfield image (left), and fluorescence images (excitation at 488 nm) before (middle) and after (right) incubation of TRPA1-expressing CHO cells with FM1-43 (10 μM). (B) Representative time course of the fluorescence signal at 488 nm (F₄₈₈). Cellular FM1-43 uptake was quantified as the persistent increase in F₄₈₈ after full washout of FM1-43 (ΔF₄₈₈). (C) Comparison of FM1-43 uptake by naïve CHO cells, cells expressing TRPA1, and TRPA1-expressing cells treated with agonist (MO, 20 μM) or antagonist (ruthenium red, 10 μM), with 50–200 cells/condition.

Figure 3

Stimulus-dependent changes in single-channel conductance. (A and B) Comparison between single-channel current traces in inside-out patches before (A) and during (B) stimulation with 20 μM MO. The pipette solution contained 5 mM MgCl₂. Current amplitudes were determined from the corresponding histograms (lower) by fitting of Gaussian functions. (C) Single-channel IV curves obtained before (black) and during (red) stimulation with MO. Lines represent linear fits forced through the origin. (D and E) Superimposed single-channel current traces obtained during voltage ramps in the cell-attached configuration before (A) and during (B) stimulation with 20 μM MO. The pipette solution contained 1 mM MgCl₂. A linear approximation of the inward and outward open-channel current level was fit by eye, yielding the indicated conductances. This experiment is representative of five similar cell-attached recordings.

Figure 4

Stimulus-dependent changes in relative Ca²⁺ permeability. (A and B) Comparison of the IV relations obtained with external solutions containing 150 mM Na⁺ or 100 mM Ca²⁺ as the sole extracellular cation, before (A) and during (B) stimulation with 20 μM MO. (C and D) Same as A and B, but for the TRPA1 pore mutant D918C. (E) P_Ca/P_Na values for WT TRPA1 and the different mutants obtained before and during stimulation with 20 μM MO. Each data point represents the mean ± SE from at least five cells.

Figure 5

Stimulus-dependent changes in relative Mg²⁺ permeability. (A and B) Comparison of the IV relations obtained with external solutions containing 150 mM Na⁺ or 100 mM Mg²⁺ as the sole extracellular cation, before (A) and during (B) stimulation with 20 μM MO. (C and D) Same as A and B, but for the TRPA1 pore mutant D918Q. (E) P_Mg/P_Na values for WT TRPA1 and the different mutants obtained before and during stimulation with 20 μM MO. Each data point represents the mean ± SE from at least five cells.

Figure 6

Fractional Ca²⁺ currents through TRPA1. (A) Representative example of the determination of Pf% for the nonstimulated WT TRPA1 current. The decrease in Fura2-fluorescence at 380 nm (in arbitrary fluorescence units (FU)) and the integrated inward current were measured during a 1-s step to −80 mV from a holding potential of +80 mV (protocol shown above), in extracellular solution containing 2 mM (left) or 100 mM (right) Ca²⁺. Using Eq. 4, this recording yielded a Pf% of 17.9%. (B) Same as A, but in a cell stimulated with 20 μM MO. Using 4, this recording yielded a Pf% of 22.3%. (C) Average Pf% values for WT and mutant TRPA1, in both nonstimulated and MO-stimulated conditions. Each data point represents the mean ± SE from at least five cells.

Figure 7

Comparison of P_Ca/P_Na and Pf% values for WT and mutant TRPA1 and for TRPV1. For TRPA1, solid symbols represent the basal state and open symbols the MO-activated state. For TRPV1, the solid and open symbols represent the current activated by pH 5 and 100 nM capsaicin, respectively (values taken from Chung et al. (24) and Samways et al. (34)). Solid line represents the prediction of the GHK-equation (Eq. 5). The dotted line represents a modified GHK equation assuming that Ca²⁺ ions provoke 35% block of the monovalent cation current.