Irritating channels: the case of TRPA1

Abstract

Transient receptor potential (TRP) channels have been extensively studied over the past years. Yet, in most cases, the gating mechanisms of these polymodal cation channels still remain a puzzle. Using the nociceptive channel TRPA1 as an example, we discuss the role of dynamic regulation of the pore size (pore dilatation) on channel gating. Additionally, we critically revise current knowledge of the role of intracellular domains, such as ankyrin repeats and EF hand motifs, in channel activation and function. Finally, we assess some problems inherent to activation of TRPA1 by the reaction of electrophilic compounds with the nucleophilic thiol sink of N-terminal reactive cysteines.

Figure 1. Structural scheme of TRPA1. Domain structure of TRPA1 monomers

The domains were elucidated by PROSITE at the ExPASy proteomics server (Swiss Institute of Bioinformatics). Pleckstrin homology (PH) refers to putative phosphoinositol phosphate binding motifs as defined previously (Nilius et al. 2008). CaM, calmodulin; EF hand indicates putative Ca²⁺ interaction sites.

Figure 2. TRPA1 single channels currents from cell-attached patches

A, cell-attached single channel recording of TRPA1 at different membrane potentials (Note the short burst of opening at positive potentials and the high open probability at negative potentials. The size of the unitary current was measured from amplitude histograms fitted by two Gaussian distributions and plotted in the i–V curve in B. B, from the single channel i–V curves, a single channel conductance under these conditions of 71 pS for negative potentials and 110 pS for positive potentials was calculated. Mean currents were obtained from averaging between 12–36 traces as shown at the left and plotted against voltage (squares). C, dividing the single amplitude of the mean current by the amplitude of the single channel currents at all measured potentials resulted in the P_open–V plot. This curve is fitted by the Boltzmann function which describes the voltage dependence of TRPA1 inactivation. The pipette contained 1.5 mm CaCl₂ and 1 mm MgCl₂. For more technical details see Karashima et al. 2007, , .

Figure 3. TRPA1 single channel currents from an inside-out patch before and after application of PPP_i and from a cell-attached patch expressing the pore mutant D918Q

A, single channel measurement from an inside-out patch immediately after excision (wild type TRAP1, pipette 0 Ca²⁺, 5 Mg²⁺, +80 mV). B, same conditions as in A. The inner site of the membrane is exposed to 5 mm PPP_i. C, cell-attached measurement of single channel currents through the less Ca²⁺-permeable D918Q mutant (pipette solution as in A, +80 mV). Note that all experiments were performed with a sampling rate of 2.5 kHz and a filtering of 1 kHz. Although it cannot be excluded that very fast openings are not completely resolved, the striking difference to the channel behaviour in A and B is obvious.