Single-Point Mutations in Ankyrin Repeat Six Make Mouse TRPA1 Sensitive to Hot Temperatures

Abstract

A subset of Transient Receptor Potential (TRP) ion channels is expressed in sensory neurons and enables thermosensation in mammals. These TRP channels are activated with high sensitivity either by hot or cold temperatures. The molecular mechanism of this temperature-directionality (heat vs. cold) is not understood. TRPA1 is special in this respect, because several TRPA1 orthologues are activated by either heat or cold or are temperature-insensitive. We hypothesized that specific amino-acid residues in TRP channels determine temperature-directionality and that mutation of these residues might shift the thermal-activation profile. To test this hypothesis, we generated a library of cold-activated mouse TRPA1 containing ∼12,000 random mutant clones and stimulated it with hot temperatures, while measuring channel activity in a FLIPR calcium assay.We discovered three single-point mutations in mouse TRPA1 that each cause strong heat-sensitivity in TRPA1. All three mutations are located in ankyrin repeat six. The sensitivity to chemical agonists remained unaffected in all three mutants. Electrophysiological characterization of one point-mutant revealed a strong temperature-dependence of voltage-sensitivity, the rate of channel activation but not deactivation, and the open-probability but not unitary conductance. Taken together, our data demonstrate that single-point mutations are sufficient to make mouse TRPA1 strongly heat-sensitive and that chemical activation can be uncoupled from temperature activation. We propose that a change in the coupling between temperature-sensing and channel-gating causes this change in temperature-directionality.