Mitochondrial ryanodine receptors and other mitochondrial Ca2+ permeable channels

Abstract

Ca(2+) channels that underlie mitochondrial Ca(2+) transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca(2+) uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca(2+) transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca(2+) overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca(2+) channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca(2+) channels.

Figure 1

Mitochondrial Ca²⁺ channels/transporters and role in mitochondrial function. Mitochondrial Ca²⁺ uptake is determined by the mitochondrial Ca²⁺ uniporter (MCU), rapid mode of uptake (RaM), and ryanodine receptor (mRyR, or RyR1). The mitochondrial permeability transition pore (mPTP), Na⁺/Ca²⁺ exchanger (mNCX), H⁺/Ca²⁺ exchanger (mHCX, encoded by Letm1), and DAG activated cation channels (DCC) contribute to Ca²⁺ efflux. Mitochondrial Ca²⁺ uptake contributes to (a) shaping cytosolic Ca²⁺ signals and triggering metabolic coupling by enhancing mitochondrial ATP synthesis: (b) stimulation of Ca²⁺ dependent dehydrogenases of the TCA cycle [34] to increase NADH/FADH production used to feed electrons through the electron transport chain (ETC) and (c) activation of the ATP synthase [35]. However, mitochondrial Ca²⁺ overload can trigger (d) mPTP activation, (e) ROS generation, and cell death. Voltage dependent anion-selective channels (VDAC) provide a pathway for Ca²⁺ and metabolite transport across the mitochondrial outer membrane. MIM and MOM; mitochondrial inner and outer membranes, respectively.

Figure 2

Ca²⁺ dependence of the major mitochondrial Ca²⁺ influx pathways. Relative activity of RaM (blue), mRyR (red), and the MCU (black) is estimated based on respective Ca²⁺ dependencies assuming a constant membrane potential and electrochemical gradient across the mitochondrial inner membrane. MCU is modeled according to patch clamp data of Kirichok et al. [43] and fitting with a Hill equation, 1/(1+(K_m/x)ⁿ), where K_m is the half-maximal concentration (19 mM) for activation, x is the extra-mitochondrial Ca²⁺ concentration, and n is the Hill coefficient (0.6). mRyR is modeled based on the Ca²⁺ dependent activation and inhibition of RyR1 channels using a modified Hill equation, c1*(1/(1+(K_a/x)ⁿ))*(1−1/(1+(K_i/x)ⁿ)), where c1 is a constant (0.0045) to enforce a 5-fold faster Ca²⁺ transport by mRyR compared to the MCU at 1 μM extra-mitochondrial Ca²⁺ according to the UV flash-induced mitochondrial Ca²⁺ uptake experiments of Beutner et al [10], K_a (2 μM) is the half-maximal concentration for Ca²⁺ dependent activation, K_i (20 μM) is the half-maximal concentration for Ca²⁺ dependent inhibition, x is the extra-mitochondrial Ca²⁺ concentration, and n is the Hill coefficient (4). RaM is modeled based on the same modified Hill’s equation, c2*(1/(1+(K_a/x)ⁿ))*(1−1/(1+(K_i/x)ⁿ)), where c2 is a constant (0.00049) to enforce a 50-fold faster Ca²⁺ transport of RaM compared to the MCU at 50 nM extra-mitochondrial Ca²⁺ according to the findings of Buntinas et al [8], K_a (20 nM) is the half-maximal concentration for Ca²⁺ dependent activation, K_i (100 nM) is half maximal concentration for Ca²⁺ dependent inhibition, x is the extra-mitochondrial Ca²⁺ concentration, and n is the Hill coefficient (4).

Figure 3

Representative single channel current traces of different mitochondrial Ca²⁺ channels. A. Single channel current traces of MiCa recorded from a COS-7 cell mitoplast reproduced from the original recording reported by Kirichok et al. [43] (Nature, 2004, 427:360–364, supplemental figure 2a) with permission from Nature Publishing Group. The single channel activity of MiCa shows multiple conductance states ranging from 2.6–5.2 pS at −160 mV in symmetrical 105 mM CaCl₂. At positive voltages (+140 mV), the single channel activity of MiCa shows fast flickering with very small subconductance states, an indication of conduction block by Ca²⁺. B. Single channel current trace of a mRyR channel purified from rat heart mitochondrial inner membrane and incorporated into an artificial lipid bilayer reproduced from the original recording reported by Altschafl et al. [12] (Biochimica et Biophysica Acta - Biomembranes, 2007, 1768:1784–1795, figure 7c) with permission from Elsevier. Peak unitary mRyR conductance is between 500–800 pS using a 300/50 mM, cis/trans, Cs-methanesulfonate gradient and 50 μM cytosolic Ca²⁺. Impera toxin A, a RyR channel modulator, induces subconductance opening of mRyR. C. Single channel current trace of a DAG activated cation channel (DCC) recorded from a brain mitoplast reproduced from the original report of Chinopoulos et al. [63] (Journal of Bioenergetics and Biomembranes, 2007, 37(4):237–247, figure 4d) with permission from Springer. The DCC current was recorded at −50 mV in symmetrical 150 mM KCl in the presence of 10 μM cytosolic Ca²⁺ and 100 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analog. D. Single channel current trace of mPTP, previously referred to as the multi-conductance channel (MCC), recorded from rat heart mitoplast reproduced from the original report of Kinnally et al. [71] (Journal of Bioenergetics and Biomembranes, 24(1):99–110, figure 3) with permission from Springer. The mPTP activity was recorded at −60 mV in symmetrical 150 mM KCl. E. Single channel current traces of VDAC reproduced from the original report of Pavlov et al. [67] (Biochimica et Biophysica Acta - Bioenergetics, 2005, 1710:96–102, figure 2b & 2c) with permission from Elsevier. (a) Typical voltage dependent channel activity of VDAC with voltage ramps between ±40 mV shows anion- or cation-selective conductance states. (b) VDAC activity was recorded at 0 mV with 150/30 mM KCl gradient. Anion- and cation- selective states are shown with arrows. Solid line indicates the 0 current level. Scale bars are presented with some modification from the original article.