Guanosine Triphosphate

Patch-clamp recordings (in whole-cell or inside-out configuration) were performed by using an EPC10 amplifier (HEKA Elektronik, Germany) controlled by PATCHMASTER. Patch pipettes were made from borosilicate glass and fire-polished to a resistance of ~3 MΩ. Both the pipette and the bathing standard solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4, adjusted with NaOH). The different pH values of bathing solution were adjusted with HCl. To record the currents evoked by increased temperatures, the membrane potential was held at −60 mV. For bi-ionic reversal potential measurements of monovalent ions, after the whole-cell configuration was obtained in standard solution, the bathing solution was changed to 140 mM NaCl (or KCl or CsCl), 10 mM HEPES, and 10 mM glucose (adjusted to pH 6.1 with NaOH, KOH, or CsOH, respectively). For divalent cation permeability experiments, the bathing solution was changed to 110 mM MgCl₂ (or CaCl₂ or BaCl₂), 2 mM Mg(OH)₂ [or Ca(OH)₂ or Ba(OH)₂], 10 mM HEPES, and 10 mM glucose, pH 6.1 (adjusted with HCl). Given that the frequent perfusion of different bathing solutions disturbed stability of temperature control, each recorded cell experienced only one exchange of bathing solution. Therefore, currents in the presence of different bathing solutions were normalized to the value at +80 mV before liquid junction potentials were corrected. A voltage ramp was employed to indicate the reversal membrane potential. Permeability ratios for monovalent cations to (P_X/P_Na) were calculated as previously described (30 (link)): P_X/P_Na = exp(ΔV_revF/RT), where V_rev presents the reversal potential, F represents Faraday’s constant, R is the universal gas constant, and T is absolute temperature. For measurements of divalent permeability, P_Y/P_Na = [Na⁺]_i exp(ΔV_revF/RT)(1 + exp(ΔV_revF/RT))/4[Y²⁺]_o, where the bracketed terms are activities. Assumed ion activity coefficients are 0.75 for monovalents and 0.25 for divalents. To record the heat activation of BRTNaC1 in the presence of physiological solution, the bathing solution contained (in mM) 120 NaCl, 5 KCl, 4 MgCl₂, 1.5 CaCl₂,10 N-Tris-(hydroxymethyl)-methyl-2-amino-ethanesulfonic acid (TES), 25 proline, and 5 alanine. Physiological pipette solution contained (in mM) 140 K gluconate, 10 TES, 4 Mg adenosine triphosphate, 2 MgCl₂, and 0.4 Na guanosine triphosphate. For inhibitor screening, the perfusion and solution switching were carried out by a gravity-driven system (RSC-200, BioLogic). The solutions flowed through separated tubes to minimize mixing of the solutions. The patches were placed at the perfusion tube outlet.
The experimental apparatus for laser irradiation was as previously reported (31 (link)), where the energy of laser photons was absorbed by water molecules and converted to thermal energy. Briefly, a controller (Thorlabs, maximal optic output power set at 300 mW) was used to drive a laser diode (Fitel), which generated the laser beam with an emission peak of 1,443 nm. The patch pipette tip was placed in front of the center of the optical fiber so that the cells or patches could be heated by different temperatures. To calibrate the relationship between laser driving power (indicated by voltage) and temperature (in °C), the temperature of bathing solution (room temperature, n = 5) and the recorded boiling points of pure ethanol or water (n = 5 for each) were used to fit this relationship using a two-point method. The data points were fitted to a linear equation that describes the relationship between laser driving power and temperature.

The cDNA library was prepared using SuperScript III reverse transcriptase (Thermo Fisher Scientific) and an oligo(dT) primer from total RNAs extracted from the Neuro-2A cells. The Mark2 3′UTR cDNA insert was ligated to a pCAG neo vector by In-Fusion HD cloning (Takara Bio) and amplified in E. coli strain DH5α to obtain a pCAG-Mark2 3′UTR plasmid. Plasmids containing G4 deletion mutants were generated using the KOD-Plus Mutagenesis Kit (Toyobo) from the pCAG-Mark2 3′ UTR plasmid. T7 promoter–containing DNA templates were prepared by a standard PCR method using the PrimeSTAR Max DNA Polymerase (Takara Bio). In vitro transcription was performed using T7 RNA polymerase (Roche) in appended transcription buffer (Roche) supplemented with 2.5 mM nucleotide triphosphates (Takara Bio), RNasin Ribonuclease Inhibitor (1 U/μl; Promega), and an 800-ng DNA template at 37°C for 6 to 8 hours. For the synthesis of 7-deazaguanosine–substituted RNA, 7-deazaguanosine-5′-triphosphate (TriLink BioTechnologies) was added to the reaction instead of guanosine 5′-triphosphate (GTP) and the Ribo m7G Cap Analog (Promega). The reaction solution was treated with deoxyribonuclease I (0.08 U/μl; Thermo Fisher Scientific) at 37°C for 15 min and purified using the RNeasy Mini Kit (Qiagen). The purified Mark2 3′UTR RNA was capped and polyadenylated using the Vaccinia Capping System (BioLabs) and E. coli Poly(A) Polymerase (BioLabs), respectively, and then labeled with CX-rhodamine using the Label IT Nucleic Acid Labeling Kit (Mirus).