Figure 1.

Bimodal activation of TRPY1. (A) A diagram showing that TRPY1 senses membrane stretch force through the membrane-embedded domains and Ca²⁺ through the cytoplasmic domains. Energetically, the open probability is governed by ΔG₀ − ΔG_force − ΔG_Ca²⁺, where ΔG₀ refers to the energy difference between the C and O state before the application of force or Ca²⁺. ΔG₀ includes the energies from structures, temperature, protonation, etc. (B) The P_o versus pressure plots at different [Ca²⁺]. For each of the three [Ca²⁺], P_o from TRPY1 ensemble currents is plotted against the applied pressure and fitted with the following Boltzmann equation: P_o = 1/[1 + a × exp(−Δα × P)], which is transformed from P_o = 1/[1 + exp((ΔE − γ × ΔA)/k_B × T)], where a and Δα are fitting parameters in the P_o–pressure plot, with Δα (in millimeters of Hg⁻¹) being the slope parameter; P (in millimeters of Hg) is the applied pressure; ΔE is the free energy of the channel at given [Ca²⁺]; g (in Newtons per meter) is the membrane tension; ΔA (in square meters) is the expansion coefficient; k_B is the Boltzmann constant; and T is the absolute temperature. The transformation assumes that the radius of our patches, r (in meters), remains constant. We strictly controlled the sizes of our pipette tips in a narrow range with the bubble numbers 4.5–5.1 in ethanol(Sakmann and Neher, 1983) to approach the constancy of r. With this assumption, γ can be converted to P according to Laplace’s law. The saturation levels of channel activities were determined by applying high pressure at high [Ca²⁺] (10⁻³ M). See Materials and methods for details. Although [Ca²⁺] laterally shifts P_o–pressure curves, all three fits have a similar Δα value (Table I), indicating that Δα defines the mechanosensitivity and is independent of [Ca²⁺]. Means ± SD (not SEM; n = 4 for 10⁻³ M, n = 6 for 10⁻⁵ M, and n = 4 for 10⁻⁶ M Ca²⁺).