The dilated TRPA1 channel pore state is blocked by amiloride and analogues

Abstract

TRPA1 channels are a member of the transient receptor potential (TRP) superfamily. Several of its members, including TRPA1 can exist in at least two distinct open states: a restricted and a dilated state. The restricted state is a tetramer non-selective cation channel, whereas the dilated state allows influx of much larger molecules, e.g. Yo-Pro (Mw~630). The exact nature of the dilated channel is not well understood, however it was recently shown that the dilated state is regulated by extracellular divalent, especially calcium. Using open channel blockers as tool compounds and a combination of calcium imaging, fluorescence dye uptake and whole-cell patch clamp recordings I here demonstrate that amiloride and its analogue 5-(N,N-Dimethyl)amiloride (DMA) block the channels at low but not at high extracellular calcium. Hence, these data suggest that amiloride and other open channel blockers bind to sites revealed during the dilation process. Furthermore, the same series of compounds blocked the agonist-induced Yo-Pro uptake in TRPA1 expressing cells. Thus, these results support the hypothesis that in low extracellular calcium the TRP channels are dilating, and as a consequence open channel blockers such as amiloride are allowed deeper into the pore providing a more efficient block. The TRP channel dilation mechanism may play important roles in many sensory processes, including pain and hearing.