Bassetto CAZ et al. (2023), Ion channel thermodynamics studied with tempera...

XB-ART-59640

Biophys J 2023 Feb 21;1224:661-671. doi: 10.1016/j.bpj.2023.01.015.

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Ion channel thermodynamics studied with temperature jumps measured at the cell membrane.

Bassetto CAZ , Pinto BI , Latorre R , Bezanilla F .

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Perturbing the temperature of a system modifies its energy landscape, thus providing a ubiquitous tool to understand biological processes. Here, we developed a framework to generate sudden temperature jumps (Tjumps) and sustained temperature steps (Tsteps) to study the temperature dependence of membrane proteins under voltage clamp while measuring the membrane temperature. Utilizing the melanin under the Xenopus laevis oocytes membrane as a photothermal transducer, we achieved short Tjumps up to 9°C in less than 1.5 ms and constant Tsteps for durations up to 150 ms. We followed the temperature at the membrane with sub-ms time resolution by measuring the time course of membrane capacitance, which is linearly related to temperature. We applied Tjumps in Kir1.1 isoform b, which reveals a highly temperature-sensitive blockage relief, and characterized the effects of Tsteps on the temperature-sensitive channels TRPM8 and TRPV1. These newly developed approaches provide a general tool to study membrane protein thermodynamics.

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Species referenced: Xenopus laevis
Genes referenced: trpm8 trpv1
GO keywords: ion channel activity [+]

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	Figure 1. Tjump and capacitance-based temperature measurement techniques. (A) Schematic representation of Tjump setup. The voltage of oocyte dome was controlled using cut-open voltage clamp and at the same time it was illuminated using a homogenized laser beam. A calibrated pipette was positioned close to the membrane to measure the temperature. (B) Equivalent circuit representation of the voltage-clamped membrane. (C) Applied sinusoidal voltage wave (blue) and elicited current (red). (D) Relationship between normalized capacitance and temperature. The capacitance was normalized for each cell by dividing its value measured at 19°C. The line represents the regression line with slope of 1.070% ± 0.003% per 1°C. (E) Capacitance change (obtained from the imaginary part of the impedance) during a laser pulse of 10 ms in duration. The duration of the laser pulse is indicated by the blue line. (F) Temperature change obtained by CTM (black) and using a calibrated pipette placed near the oocyte membrane (light blue).
	Figure 2. Effects of Tjumps on Kir ionic currents. (A) Currents induced on Kir by 10 ms Tjumps at different voltages. Inset is the voltage protocol. The temperature change measured by CTM is shown in blue, and the arrow indicates when the Tjumps were started. The dashed lines indicate isochrones used to obtain the I-V curves. (B) Normalized currents (I/ImaxT0) versus voltage for the different isochrones and their respective temperature (T0 ∼ 13°C). (C) G-V relationship of Kir for different temperatures. The G-Vs at different temperatures were fitted using Eq. 3 with a shared slope (z = 1.25) for all the curves. (D) Temperature-dependence of Vr1/2 obtained from the G-V fits in (C). The experimental values were fitted using Eq. 4 described in the materials and methods. The fitted values were ΔS = −263 ± 10 cal/K.mol and ΔH = −71.7 ± 2.9 kcal/mol. Data are represented as the mean ± standard deviation (n = 3).
	Figure 3. Using the pulse width modulation (PWM) of the laser to build up Tsteps. (A) Modulation waveform utilized to obtain a Tstep. Insets show an expanded time window to appreciate better the PWM pulses used. Red represents the beginning and blue for the end of the PWM pulses. (B) Optocapacitive current elicited by Tstep at a holding potential of −100 mV. (C) Change in temperature measured by CTM method. Insets are the expanded time window for the rising phase of temperature (red) and the Tstep end (blue). To see this figure in color, go online.
	Figure 4. Effects of Tsteps on TRPM8 and TRPV1. (A) and (B) are, respectively, the representative current traces for TRPM8 and TRPV1. Top panel shows the Tstep for each case. Middle panel shows the current elicited under Tstep, and bottom is the current without Tstep. Inset shows the voltage protocol used to elicit the currents. The arrow indicates the time where the temperature step was applied. The blue and red square indicate the time at which the currents were taken to obtain the I-V curves. The time and current scales are the same for (A) and (B). Please note that the time scales for Tstep and recorded currents are different. (C) and (D) are the I-V relationship for TRPM8 and TRPV1 ionic currents, respectively. The I-Vs were normalized by the maximum current recorded in absence of a Tstep. Data are represented as the mean ± standard deviation for experiments in bath temperature condition (n = 4). To see this figure in color, go online.
	Figure 5. Time-dependence effects of Tstep on TRPM8 and TRPV1 ionic currents. (A) and (B) are ionic currents traces for TRPM8 and TRPV1, respectively. The temperature pulse is applied when the currents reach steady state, as indicated by the arrow. Inset is the voltage protocol and Tstep profile. Please note that the time scales for Tstep and recorded currents are different. (C) Biphasic behavior of currents elicited by Tsteps for TRPM8 at 150 mV and TRPV1 at 120 mV. The arrows indicate changes associated with Δγ and ΔPO. (D) Tsteps applied at different times during a voltage protocol for TRPM8 and TRV1. Arrows indicate different times where a Tstep was applied. To see this figure in color, go online.

References [+] :

Baez, Gating of thermally activated channels. 2014, Pubmed