Hypotonic Solutions

Patch-clamp experiments using mitoplasts were performed as described previously [18] (link), [19] (link). Briefly, mitoplasts were prepared from a sample of human astrocytoma mitochondria placed in a hypotonic solution (5 mM HEPES, 200 µM CaCl₂, pH = 7.2) for approximately 1 min to induce swelling and breakage of the outer membrane. Then, a hypertonic solution (750 mM KCl, 30 mM HEPES, 200 µM CaCl₂, pH = 7.2) was added to restore the isotonicity of the medium. The patch-clamp pipette was filled with an isotonic solution containing 150 mM KCl, 10 mM HEPES, and 200 µM CaCl₂ at pH = 7.2. Mitoplasts are easily recognizable due to their size, round shape, transparency, and presence of a “cap”, characteristics that distinguish these structures from the cellular debris that is also present in the preparation. An isotonic solution containing 200 µM CaCl₂ was used as the control solution for all of the presented data. The low-calcium solution (1 µM CaCl₂) contained the following: 150 mM KCl, 10 mM HEPES, 1 mM EGTA and 0.752 mM CaCl₂ at pH = 7.2. All of the modulators of the channels and the substrates and inhibitors of the respiratory chain were added as dilutions in isotonic solution containing 200 µM CaCl₂. To apply these substances, we used a perfusion system containing a holder with a glass tube (made in our workshop), a peristaltic pump, and Teflon tubing. The mitoplasts at the tip of the measuring pipette were transferred into the openings of a multibarrel “sewer pipe” system in which their outer faces were rinsed with the test solutions (Fig. 1A). The configuration of our patch-clamp mode is presented in Fig. 1A. The experiments were carried out in patch-clamp inside-out mode. This is based on observations with various mitochondrial substrates applied such as NADH or succinate. Reported voltages are those applied to the patch-clamp pipette interior. Hence, positive potentials represent the physiological polarization of the inner mitochondrial membrane (outside positive).
The electrical connection was made using Ag/AgCl electrodes and an agar salt bridge (3 M KCl) as the ground electrode. The current was recorded using a patch-clamp amplifier (Axopatch 200B, Molecular Devices Corporation, USA). The pipettes, made of borosilicate glass, had a resistance of 10–20 MΩ and were pulled using a Flaming/Brown puller.
The currents were low-pass filtered at 1 kHz and sampled at a frequency of 100 kHz. The traces of the experiments were recorded in single-channel mode. The illustrated channel recordings are representative of the most frequently observed conductance for the given condition. The conductance of the channel was calculated from the current-voltage relationship (data not shown). The probability of channel opening (P_o, open probability) was determined using the single-channel search mode of the Clampfit 10.2 software. Calculations were performed using segments of continuous recordings lasting 60 s, with N>1000 events. Data from the experiments are reported as the mean values ± standard deviations (S.D.). Student’s t-test was used for statistical analysis. In figures showing single-channel recordings, “-” indicates the closed state of the channel.

HeLa cells were transfected with mitochondrial Aequorin WT (mtAEQ WT). After 48 h, cells were incubated with 5 μM coelenterazine for 1.5 h in Krebs–Ringer modified buffer (KRB: 125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgSO4, 5.5 mM glucose, and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4, at 37°C) supplemented with 1 mM CaCl₂, and then transferred to the perfusion chamber.
Aequorin measurements were acquired in KRB supplemented with 1 mM CaCl₂, and the agonist was added to the same medium as indicated in figure legends. Cells were then lysed with Triton X-100 in a hypotonic Ca²⁺-rich solution (10 mM CaCl₂ in H2O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca²⁺] values using the algorithm reported in Rizzuto et al. (1992) (link).

Leukocyte was isolated according to the method of O’Loughlin et al [12 (link)]. Briefly, blood leukocytes were isolated using a hypotonic solution and a hypertonic solution. The leukocyte pellets were suspended in 1 mL of TRI Reagent® solution (TRIzol) (Sigma-Aldrich Ireland Ltd., Dublin, Ireland), and stored in a sterile tube at −70°C until RNA isolation.