C-terminal acidic cluster is involved in Ca2+-induced regulation of human transient receptor potential ankyrin 1 channel

Abstract

The transient receptor potential ankyrin 1 (TRPA1) channel is a Ca(2+)-permeable cation channel whose activation results from a complex synergy between distinct activation sites, one of which is especially important for determining its sensitivity to chemical, voltage and cold stimuli. From the cytoplasmic side, TRPA1 is critically regulated by Ca(2+) ions, and this mechanism represents a self-modulating feedback loop that first augments and then inhibits the initial activation. We investigated the contribution of the cluster of acidic residues in the distal C terminus of TRPA1 in these processes using mutagenesis, whole cell electrophysiology, and molecular dynamics simulations and found that the neutralization of four conserved residues, namely Glu(1077) and Asp(1080)-Asp(1082) in human TRPA1, had strong effects on the Ca(2+)- and voltage-dependent potentiation and/or inactivation of agonist-induced responses. The surprising finding was that truncation of the C terminus by only 20 residues selectively slowed down the Ca(2+)-dependent inactivation 2.9-fold without affecting other functional parameters. Our findings identify the conserved acidic motif in the C terminus that is actively involved in TRPA1 regulation by Ca(2+).

FIGURE 1.

Truncations in C terminus reveal region involved in Ca²⁺-dependent inactivation. A, alignment of distal C terminus of TRPA1 from various species. The predicted secondary structure for hTRPA1 is indicated above the alignment. The region of interest is boxed. The residues in human TRPA1 that were mutated in this study are indicated in bold type. B, time course of representative whole cell currents through human TRPA1 measured at +80 mV and −80 mV as marked. The application of 100 μm Cin and subsequent addition of 2 mm Ca²⁺ are indicated above. The right panel shows current-voltage relationships of traces measured at times indicated by a, b, and c. C and D, voltage-ramp protocol as in B used for truncation mutants. Note the obviously slower inactivation of the TRPA1-Δ20 truncation mutant upon the addition of 2 mm Ca²⁺ to the bath solution compared with WT in B. E, average rate of current decay represented as T₅₀ for wild-type TRPA1 and truncation mutant TRPA1-Δ20. To obtain a similar rate of inactivation to the wild-type TRPA1, a 5-fold higher concentration of calcium needed to be introduced for the TRPA1-Δ20 mutant. *, p = 0.006, Student's t test. The data represent the means ± S.E.; n ≥ 6 for wild type and n ≥ 3 for mutant.

FIGURE 2.

Mutations in C-terminal acidic region alter voltage and cinnamaldehyde sensitivity of TRPA1. A, left bar graph depicts average TRPA1 currents at +80 mV in Ca²⁺-free extracellular solution before (control) and after 40 s of 100 μm Cin exposure. The right bar graph indicates relative activation by Cin for wild-type channel and individual mutants. The data represent the means ± S.E.; n ≥ 6. The asterisks indicate significant differences between mutant and wild-type TRPA1. *, p < 0.05, unpaired t test. Broken vertical lines indicate the mean values obtained from wild-type TRPA1. B, average currents at +80 mV for wild-type TRPA1 and gain of function mutants E1077A and E1077K. The horizontal bars above the records indicate the duration of Cin and Ca²⁺ application. Note the difference in basal activation level at the very beginning of the record. The data represent the means ± S.E. for n ≥ 6. C, representative current traces in response to indicated voltage step protocol (holding potential, −70 mV; voltage steps from −80 to +200 mV; increment +20 mV), recorded ∼1 min after whole cell formation. The bath solution contained 160 mm NaCl, 2.5 mm KCl, 1 mm CaCl₂, 2 mm MgCl₂, 10 mm HEPES, 10 mm glucose. D, average conductances obtained from voltage step protocols as in C. The data represent the means ± S.E.; n ≥ 8.

FIGURE 3.

Potentiation of TRPA1 mutants by extracellular Ca²⁺. A, average TRPA1 currents evoked in response to 40 s of exposure to 100 μm Cin and subsequent addition of 2 mm Ca²⁺ as indicated by horizontal bars. The average current for the wild type is shown for comparison in gray in each plot. The currents are normalized to their maximal cinnamaldehyde responses obtained prior to the addition of Ca²⁺ to the bath solution. The data represent the means ± S.E. for the number of cells indicated. For D1080A, the average current for nonpotentiated cells (in red) is overlaid onto the average current from all cells. B, average data from experiments as in A. Calcium-induced potentiation was measured as the fold increase in current, measured at +80 mV, following the addition of 2 mm (left bar graph) or 10 mm (right bar graph) extracellular Ca²⁺. The asterisks indicate significant differences between mutant and wild-type TRPA1. *, p < 0.05, unpaired t test. The lower bar graph represents the probabilities obtained from the t tests that compared the individual mutants with the wild type. C, distribution of time to peak of Ca²⁺-induced potentiation for wild-type TRPA1 channels. For 2 mm Ca²⁺, four cells had time-to-peak outside the range of 60 s. D, D1080A mutant of hTRPA1 expresses on the surface of HEK293T cells to a similar level as a control molecule containing membrane-targeting motif. Panel a, typical confocal image from cell expressing D1080A mutant of the C-terminally GFP-tagged human TRPA1 and a fluorescence intensity profile plotted for the cross-section indicated above. Panel b, wide-field fluorescence image of HEK293T cell expressing D1080A mutant of the C-terminally GFP-tagged human TRPA1. The excitation wavelength was 470 nm, and emission was detected at 530 nm. Below is a fluorescence intensity profile plotted for the cell and a cross-section (marked by line) indicated above. Panel c, wide field fluorescence image of a fluorescent marker for the membrane surface, cyan fluorescent protein-tagged pleckstrin homology domain of phospholipase Cδ1 (CFP-PH), expressed in another HEK293T cell for comparison. Below, typical fluorescence intensity profile plotted for the cell and cross-section indicated above. The excitation wavelength was 430 nm, and emission was detected at 475 nm.

FIGURE 4.

Mutations in C-terminal acidic domain affect kinetics of Ca²⁺-induced potentiation. A, 10–90% rise time of Ca²⁺-induced potentiation (2 mm) for wild-type and mutant TRPA1. The broken vertical line indicates the mean value obtained from wild-type TRPA1. B, average data from experiments as in Fig. 3A. Inactivation was measured at +80 mV and quantified as the time, relative to the peak, at which the currents had decayed to 50% of their maximum value. The broken horizontal lines indicate the mean values obtained from wild-type TRPA1 for 2 and 10 mm Ca²⁺. In A and B, asterisks indicate significant differences between mutant and wild-type TRPA1. *, p < 0.05, unpaired t test. C, left panel, increasing the concentration of extracellular Ca²⁺ from 2 mm to 10 mm partially restored the Ca²⁺-induced potentiation in D1080A. Right panel, increasing the intracellular Ca²⁺ concentration from 150 nm to 100 μm restored the potentiation of the cinnamaldehyde-induced currents in the D1080A mutant channels. The average current for the wild type is shown for comparison in gray in each plot. The currents are normalized to their maximal cinnamaldehyde responses obtained prior to the addition of Ca²⁺ to the bath solution. The data represent the means ± S.E. for the number of cells indicated. D, average data from experiments as in C, quantified as in Fig. 3B. The asterisk indicates a significant difference from wild-type TRPA1 measured with the high buffer internal solution containing 5 mm EGTA in the patch pipette. *, p = 0.01, unpaired t test.

FIGURE 5.

Homology modeling and molecular dynamics simulations of acidic region from TRPA1 based on Ca²⁺ activation apparatus of human BK channel (hSlo1; Protein Data Bank entry code 3MT5) as template protein. A, alignment of Ca²⁺-binding domain of BK with C-terminal acidic region from human TRPA1. B, illustration of calcium-binding site in hSlo1-TRPA1 chimera with surrounding structures. Residues from the TRPA1 protein are shown in ball and stick representation. Snapshot is the frame at 200 ns of the simulation. C, RMSD of protein and calcium-binding site. C also indicates the displacement of the calcium ion inside of the calcium-binding domain. A significant increase in RMSD at the end of simulation was identified to be caused by loose ends of the polypeptides at residues 833–869, whose structure is not known and was missing from the model structure (see “Materials and Methods”). The system was simulated for a total of 200 ns after equilibration. The time course of RMSD indicated that the system was sufficiently relaxed after 70 ns, and the calcium-binding motif was sufficiently relaxed after 110 ns. The displacement of the calcium ion was less than 1 Å for most of the simulation, indicating that the ion was stable. D, number of contacts with Ca²⁺ ion throughout simulation. E, lengths of ionic bonds between calcium ion and atoms in calcium-binding site.

FIGURE 6.

Mutations in C-terminal acidic region. A, molecular dynamics simulations of in silico alanine mutations of the BK/TRPA1 chimera. RMSDs of whole protein, calcium-binding site, and displacement of calcium ion in the predicted calcium-binding site for each mutant. Calcium ion was stable in E1079A and D1081A. Disruptions of the calcium-binding pocket were observed in D1080A and in D1082A after 11 and 4 ns of molecular dynamics simulation. The simulation of D1082A (D897A) mutation was particularly unstable, and the calcium ion left the binding site very soon after simulation entered production run. This simulation was interrupted after 10 ns of run. B, potentiation of the TRPA1 mutants D1080I and D1082I by extracellular Ca²⁺. Average whole cell currents evoked in response to 40 s of exposure to 100 μm Cin and subsequent addition of 2 mm Ca²⁺ as is indicated by horizontal bars. The average current for the wild type is shown for comparison in gray in each plot. The currents are normalized to their maximal cinnamaldehyde responses obtained prior to the addition of Ca²⁺ to the bath solution. The data represent the means ± S.E. for the number of cells indicated. For D1080I, the average current for nonpotentiated cells (in red) is overlaid onto the average current from all cells (in green).