Patch-clamp technique to study mitochondrial membrane biophysics

Abstract

Mitochondria are double-membrane organelles crucial for oxidative phosphorylation, enabling efficient ATP synthesis by eukaryotic cells. Both of the membranes, the highly selective inner mitochondrial membrane (IMM) and a relatively porous outer membrane (OMM), harbor a number of integral membrane proteins that help in the transport of biological molecules. These transporters are especially enriched in the IMM, where they help maintain transmembrane gradients for H+, K+, Ca2+, PO43-, and metabolites like ADP/ATP, citrate, etc. Impaired activity of these transporters can affect the efficiency of energy-transducing processes and can alter cellular redox state, leading to activation of cell-death pathways or metabolic syndromes in vivo. Although several methodologies are available to study ion flux through membrane proteins, the patch-clamp technique remains the gold standard for quantitatively analyzing electrogenic ion exchange across membranes. Direct patch-clamp recordings of mitoplasts (mitochondria devoid of outer membrane) in different modes, such as whole-mitoplast or excised-patch mode, allow researchers the opportunity to study the biophysics of mitochondrial transporters in the native membrane, in real time, in isolation from other fluxes or confounding factors due to changes in ion gradients, pH, or mitochondrial potential (ΔΨ). Here, we summarize the use of patch clamp to investigate several membrane proteins of mitochondria. We demonstrate how this technique can be reliably applied to record whole-mitoplast Ca2+ currents mediated via mitochondrial calcium uniporter or H+ currents mediated by uncoupling protein 1 and discuss critical considerations while recording currents from these small vesicles of the IMM (mitoplast diameter = 2-5 µm).

Figure 1.

Interorganelle connections of mitochondria and a schematic of the MCUcx. (A) Several Ca²⁺ channels and carriers mediate interorganelle Ca²⁺ transfer and regulation of cytosolic Ca²⁺ homeostasis. (B) Subunit composition of the MCUcx. IP₃: inositol 1,4,5-triphosphate receptor; RYR: ryanodine receptor; SERCA: sarcoendoplasmic reticulum Ca²⁺-ATPase; TRPML1: transient receptor potential mucolipin channel 1; NCLX: Na⁺/Ca²⁺/Li⁺ exchanger; CL: cardiolipin.

Figure 2.

Different steps of the whole-mitoplast patch clamp. (A) Mitochondria are isolated using differential centrifugation, and a low-pressure French press is used to break the OMM. Cartoons depict an eight-shaped and an O-shaped mitoplast with varying amounts of OMM still attached. (B) Comparison of pipette shape and tip size used for whole-mitoplast recordings with the pipette used for whole-cell patch clamp. (C and D) Mitoplast-attached (C) and the whole-mitoplast configuration (D) of the patch-clamp technique. Inset shows a DIC image of the mitoplast before and after forming the whole-mitoplast seal.

Figure 3.

Transmission electron micrographs of isolated mitochondria and mitoplast preparations. (A and B) Isolated mitochondria (A) and mitoplasts (B) from the same sample of mouse cardiac tissue. The red arrow shows an eight-shaped mitoplast and the blue arrow shows a pear-shaped mitoplast pointing toward the cap of OMM. One of each mitoplast type is demarcated with dashed lines.

Figure 4.

Schematics for the patch-clamp circuitry and current responses to seal-test protocol at different stages of seal formation. (A) A schematic of the amplifier in the whole-mitoplast voltage clamp mode. Cation currents (I_Ca2+ or I_H+) are inward when the matrix-side potential is negative. (B) An equivalent circuit for the whole-mitoplast mode. (C–F) Currents in response to −10 mV voltage steps (seal-test protocol) when recording pipette is (C) in KCl bath solution, (D) in mitoplast-attached mode with stray capacitance transients visible, (E) after stray capacitance compensation, and (F) in the whole-mitoplast configuration. R_a: access resistance (the resistance between the pipette and the mitoplast matrix); V_m: membrane potential; C_m: membrane capacitance; V_p: mitoplast potential; R_m: membrane or input resistance.

Figure 5.

Patch-clamp recording of MCU-mediated Ca²⁺ currents (I_MCU) in mitoplasts from cell lines and native tissues of mouse. (A–C) Effect of different pipette solution compositions on I_MCU in cell lines. (A) Inward current (I_MCU) density measured at −156 mV using pipette solutions with diverse compositions in MEFs and HEK293T. Each point on the bar graph represents current density from an individual mitoplast. Pipette solution composition in mM, Sol A: 110 Na⁺ gluconate, 40 HEPES, 10 EGTA, and 2 MgCl₂ (pH 7.0 with NaOH); Sol B: 110 Na⁺ gluconate, 40 HEPES, 1.5 EGTA, and 2 MgCl₂ (pH 7.0 with Tris base); Sol C: 110 Na⁺ gluconate, 40 HEPES, 1 EGTA, 5 EDTA, and 2 NaCl (pH 7.0 with Tris base); and Sol D: 130 TMA, 100 mM HEPES, 1 EGTA, 1 EDTA, and 3 NaCl (pH 7.0 with D-gluconic acid). For HEK293T, pipette solution contained (in mM) 110 Na⁺ gluconate, 40 HEPES, 5 EGTA, and 2 MgCl₂ (pH 7.0 with NaOH). Part of the data is taken from Garg et al. (2021) (Creative Commons Attribution license, also known as a CC-BY license). Data are presented as mean ± SEM. (B and C) Exemplary traces depict I_MCU elicited by 1 mM [Ca²⁺] (red) in MEF mitoplasts using either Na⁺ gluconate (B) or TMA (C) in pipette solution. Baseline currents are measured in nominally Ca²⁺-free bath solution containing 1 mM EGTA (black). Baseline currents provide a good estimation for any leaks currents that are subtracted from Ca²⁺-induced currents for I_MCU quantification. Arrows mark the outward Na⁺ currents in the positive voltage range. Voltage protocol is indicated on the top (B). (D and E) Exemplary [Ca²⁺]-dependent current responses from (D) WT and (E) MCU-KO mitoplasts from MEFs. Scale bar is the same in B–E. (F) Exemplary current responses from a WT-MEF mitoplast in response to voltage steps, in the presence of 1 mM [Ca²⁺]. Step voltage protocol is shown on the top. (G and H) Exemplary I_MCU traces from mouse (G) heart and (H) kidney, in response to 1 mM [Ca²⁺]. Scale bar is same in G and H. The voltage ramp protocol (indicated on the top of B) is the same for the current trace in B–E, G, and H. Arrows mark the outward Na⁺ currents observed only in the presence of EGTA. The dashed line in each current trace marks the zero current level.

Figure 6.

UCP1-mediated I_H+ from brown fat mitoplasts. Exemplary control trace (I_H+, red) from mouse brown fat in the presence of HEPES/EGTA solution and after the addition of 1 mM GDP (black). Step voltage protocol is shown on the top. The dashed line marks the zero current level.