Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells

Abstract

Voltage-gated K(+) (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents (I(Kv)) revealed the presence of a mitochondria-mediated Mg(2+) and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.

Fig. 1.

Kv channel currents recorded from freshly isolated rat small pulmonary arterial smooth muscle cells (PASMC). A: a representative current trace recorded using the activation protocol (top). B: current-voltage (I-V) relationship for activation derived from the tail current measured from the example shown in A. Solid lines represent the fit to the Boltzmann equation with the half-activation potential (dashed line) mV and the slope factor equal to −16.9 and 8.8 mV, respectively. Cell membrane capacitance (C_m) was equal to 14.6 pF.

Fig. 2.

Inhibition of the mitochondrial electron transport chain (mETC) shifts I_Kv activation to more negative potentials. A: schematic depicting the mETC. Sites of inhibition with specific inhibitors are indicated. B: normalized I_Kv(tail) activation in control and in the presence of 1 μM antimycin A (n = 9). I_Kv(tail) was measured at 2–3 ms after a test potential, normalized and fitted to the Boltzmann equation as described in materials and methods. Solid lines were drawn with the half-activation potentials equal to −11.3 and −22 mV (dashed lines) and the slope factors equal to 10.8 and 10.4 mV for control and antimycin A, respectively. C: change in the half-activation potential between control and in the presence of the indicated inhibitor (n = 22, 10, 9, and 14, respectively). ***P < 0.001.

Fig. 3.

Inhibition of the mETC decreases I_Kv amplitude at positive potentials. A: representative paired current traces at +50 mV for control (gray and marked as C) and in the presence of the indicated mETC inhibitors (black and marked as Rot, Myx, Ant, and cyanide for rotenone, myxothiazol, antimycin A, and NaCN, respectively). Cell capacitance was equal to 11.1 pF (rotenone), 14.2 pF (myxothiazol), 12.6 pF (antimycin A), and 14.7 pF (NaCN). Vertical and horizontal bars are equal to 1 nA and 100 ms, respectively. B: I_Kv current density in control and in the presence of 1 μM antimycin A (n = 9). C: decrease in the Kv current amplitude at +50 mV after 5-min perfusion with the indicated inhibitor (n = 22, 10, 9, and 14, respectively). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4.

Dissociation of the mitochondrial proton gradient with CCCP mimics the dual effect of the mitochondrial inhibitors. CCCP shifts I_Kv activation to more negative potentials and decreases the current amplitude at positive potentials. A: normalized I_Kv(tail) in control and in the presence of CCCP (left) and the average negative shift in the half-activation potential (right, n = 20). Solid lines were drawn in accordance with the Boltzmann equation with the half-activation potentials equal to −13.7 and −22.6 mV (dashed lines) and the slope factors equal to 9.9 and 10.0 mV for control and CCCP, respectively. B: representative current trace at +50 mV in control (gray) and in the presence of CCCP (black, left) and the average changes in the current amplitude at +50 mV (right, n = 20). C_m = 8.8 pF. **P < 0.01.

Fig. 5.

The effect of antimycin A on the negative shift in I_Kv activation is specific to its inhibition of the mETC. Proximal mETC inhibitors rotenone and myxothiazol (1 μM) and the mitochondrial uncoupler CCCP (2 μM) inhibit the antimycin A-induced (gray) negative shift in I_Kv half-activation in 8, 7, and 8 PASMCs, respectively. Cells were preincubated with each inhibitor for 5 min before recording the “control” I-V. Cells were then superfused with 1 μM antimycin A for 5 min before recording the test I-V. Experiments were performed in the whole cell configuration. The significance of the inhibition of the antimycin-induced change in half-activation is represented by the gray bar. ##P < 0.01, ###P < 0.001.

Fig. 6.

Antimycin A slowly and reversibly depolarizes PASMCs. A: representative recording of the cell membrane potential in the current clamp mode. Cell capacitance was equal to 12.3 pF. B: summary of the antimycin A-induced changes in the cell membrane potential in 3 PASMCs. *P < 0.05 compared with control.

Fig. 7.

Inhibition of the ATP transporter with oligomycin mimics the effects of the inhibition of the mETC on I_Kv activation and amplitude. A: schematic depicting the mitochondrial electron transport chain including ATP production by complex V. Sites of inhibition with specific inhibitors are indicated. B: effect of oligomycin (gray) on the half-activation (left, n = 12) and I_Kv amplitude at +50 mV (right, n = 12). Dialysis of the cells with ATP (5 mM Na₂ATP in the pipette solution) inhibited the changes in I_Kv (black) (n = 6). ##P < 0.01, indicating significance of the inhibitory effect of Na₂ATP compared with the control (gray bars).

Fig. 8.

The effects of antimycin and oligomycin on I_Kv are additive. Effect of preincubation with antimycin A on the oligomycin-induced changes in half-activation potential and the decrease in current amplitude (black columns, n = 7). For successive treatment, cells were perfused with antimycin A for 5 min and a control I-V was recorded. Cells were then perfused with antimycin A and oligomycin for 5 min before recording a “test” I-V. Oligomycin-induced changes in the presence of antimycin A was measured as the difference between the test and control recordings. Gray columns show the effects of oligomycin alone for comparison (n = 12). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 9.

Changes in intracellular magnesium concentration also contribute to the effects of the mETC inhibition on I_Kv. Effect of intracellular Na₂ATP (n = 8), MgATP (n = 9), and EDTA (n = 8) (no MgCl₂ was added to the pipette solutions) on antimycin A induced changes in half-activation potential and a decrease in I_Kv amplitude at +50 mV (A). The effect of antimycin A under control conditions is shown in gray for comparison. B: absolute half-activation potential values due to change in each pipette solution as indicated (n = 184, 9, 22, and 25, respectively). #P < 0.05, ##P < 0.01, and ###P < 0.001. #Significance of the inhibitory effect in each pipette solution compared with the control.

Fig. 10.

Inhibition of the mETC increases [Mg²⁺]_i in single PASMCs. A: representative transmitted light (left) and fluorescence images of a PASMC loaded with MagFluo-4 in response to 2 μM CCCP at the time of application indicated above. B: time dependence of changes in the intensity of the MagFluo-4 fluorescence expressed as F/F₀. Arrows indicate time points where images shown in A were taken. C: averaged effect of CCCP and antimycin A on [Mg²⁺]_i at 5- and 10-min period (n = 7 and 7, respectively). *P < 0.05, **P < 0.01.