Characterization of distinct single-channel properties of Ca²⁺ inward currents in mitochondria

Abstract

Previous studies have demonstrated several molecularly distinct players involved in mitochondrial Ca(2+) uptake. In the present study, electrophysiological recordings on mitoplasts that were isolated from HeLa cells were performed in order to biophysically and pharmacologically characterize Ca(2+) currents across the inner mitochondrial membrane. In mitoplast-attached configuration with 105 mM Ca(2+) as a charge carrier, three distinct channel conductances of 11, 23, and 80 pS were observed. All types of mitochondrial currents were voltage-dependent and essentially depended on the presence of Ca(2+) in the pipette. The 23 pS channel exhibited burst kinetics. Though all channels were sensitive to ruthenium red, their sensitivity was different. The 11 and 23 pS channels exhibited a lower sensitivity to ruthenium red than the 80 pS channel. The activities of all channels persisted in the presence of cylosporin A, CGP 37187, various K(+)-channel inhibitors, and Cl(-) channel blockers disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate and niflumic acid. Collectively, our data identified multiple conductances of Ca(2+) currents in mitoplasts isolated from HeLa cells, thus challenging the dogma of only one unique mitochondrial Ca(2+) uniporter.

Fig. 1

Time course of mitochondria swelling in hypotonic solution. Isolated mitochondria were suspended in hypotonic solution (5 mM HEPES, 5 mM sucrose, and 1 mM EGTA pH adjusted to 7.4 with KOH) and volume change due to mitochondrial swelling was monitored as decline in absorbance at 540 nm. Data are expressed as ratio between the change in absorbance over time divided by the absorbance at the initial timepoint (mean ± SD, n = 3)

Fig. 2

i-MCC are present in mitoplasts isolated from HeLa cells. a Exemplary traces of i-MCC from HeLa mitochondria at test potential of −100 mV. b Corresponding amplitude histogram constructed from traces shown in (a). c i-MCC channel activities at different voltages indicated. d Corresponding IV curve of the channel activity shown in (c)

Fig. 3

xl-MCC are present in mitochondria from HeLa cells. a Representative tracings of single-channel events at a test potential of −100 mV showing activity of xl-MCC. b Corresponding amplitude histogram constructed from traces shown in (a). c Average of individual traces shown in (a). d An exemplary recording from HeLa mitoplast showing coexistence of channels with bursting activity, i-MCC and xl-MCC in the same patch

Fig. 4

Voltage dependency of extra-large conductance Ca²⁺ channel. a Representative current traces in response to voltage ramps from −150 to +50 mV showing single-channel activity of xl-MCC. The figure depicts the net current obtained after subtraction of the background current obtained from the same patch during nonresponsive sweeps from the xl-MCC current responses. b xl-MCC activity at different voltages indicated

Fig. 5

Channels with b-MCC isolated from HeLa cells. a Exemplary traces showing the activity of intermediate conductance mitochondrial Ca²⁺ channels together with bursting channel activity in the same patch. b Exemplary traces showing the activity of bursting channel as a sole single-channel activity type. c Corresponding amplitude histogram constructed from traces shown in (b)

Fig. 6

Bursting Ca channels (b-MCC) are voltage dependent. a Exemplary single-channel traces of b-MCC at different voltages indicated. Note an increase in the b-MCC activity at more negative voltages. b Corresponding current–voltage relationship of i-MCC

Fig. 7

Pharmacological inhibition of mitochondrial Ca²⁺ channels by ruthenium red (RuR). a Exemplary traces showing a decreased unitary amplitude of xl-MCC channels in the presence of 1 μM RuR in the pipette. Test potential is −100 mV. b Exemplary traces showing reduced single-channel amplitude of i-MCC channel activity by 10 μM RuR present in the pipette solution. Standard 105 mM CaCl₂-containing solutions was supplemented with 100 μM DIDS, 10 μM CsA, 10 μM CGP 37187, and 10 μM RuR. Test potential is −100 mV. c Corresponding amplitude histogram of the channel activity shown in (b) reveals decreased single-channel amplitude of i-MCC in the presence of RuR

Fig. 8

Ruthenium red suppresses the activities of mitochondrial Ca²⁺ channels. a, b Representative traces showing the activities of b-MCC at test potential of −150 mV (a) and xl-MCC at test potential of −100 mV (b) before (control) and after addition of 1 and 10 μM RuR into the bath solution. Recordings were performed in the bath solution containing 40 mM Cl⁻ in the absence (a) and presence (b) of 100 μM DIDS in the pipette solution

Fig. 9

Effect of manipulation of Cl⁻ concentrations on (Ca²⁺) inward currents in mitoplasts. a Representative traces of i-MCC channel activity recorded with the use of low Cl⁻ (40 mM Cl⁻)-containing pipette solution. Test potential is −100 mV. b Amplitude histogram of the channel activity shown in (a). c Representative traces showing the activity of b-MCC at different voltages indicated in the presence of 40 mM bath Cl⁻. d Corresponding voltage dependency of single-channel amplitudes of single-channel openings shown in (c). e Representative current responses to a voltage ramps from −150 to +150 mV under our standard recording conditions showing the activity of Cl⁻ channels at positive potentials

Fig. 10

Pharmacological characterization of (Ca²⁺) inward currents in mitoplasts. a Representative traces showing xl-MCC channel activity in the presence of 100 μM niflumic acid in the pipette solution. Test potential is −100 mV. b Representative traces showing the activities of b-MCC and i-MCC in the same patch in the presence of 100 μM DIDS in the pipette solution. Test potential is −100 mV. c Representative traces showing the activity of b i-MCC in the presence of 1 μM paxilline and 10 μM glibenclamide in the pipette solution. Test potential is −100 mV