ATP-sensitive Potassium Channel in Mitochondria of the Eukaryotic Microorganism Acanthamoeba castellanii

Anna Kicinska,Aleksandra Swida,Piotr Bednarczyk,Izabela Koszela-Piotrowska,Katarzyna Choma,Krzysztof Dolowy,Adam Szewczyk,Wieslawa Jarmuszkiewicz

doi:10.1074/jbc.m701496200

Abstract

We describe the existence of a potassium ion transport mechanism in the mitochondrial inner membrane of a lower eukaryotic organism, Acanthamoeba castellanii. We found that substances known to modulate potassium channel activity influenced the bioenergetics of A. castellanii mitochondria. In isolated mitochondria, the rate of resting respiration is increased by about 10% in response to potassium channel openers, i.e. diazoxide and BMS-191095, during succinate-, malate-, or NADH-sustained respiration. This effect is strictly dependent on the presence of potassium ions in an incubation medium and is reversed by glibenclamide (a potassium channel blocker). Diazoxide and BMS-191095 also caused a slight but statistically significant depolarization of mitochondrial membrane potential (measured with a TPP(+)-specific electrode), regardless of the respiratory substrate used. The resulting steady state value of membrane potential was restored after treatment with glibenclamide or 1 mM ATP. Additionally, the electrophysiological properties of potassium channels present in the A. castellanii inner mitochondrial membrane are described in the reconstituted system, using black lipid membranes. Conductance from 90 +/- 7 to 166 +/- 10 picosiemens, inhibition by 1 mM ATP/Mg(2+) or glibenclamide, and activation by diazoxide were observed. These results suggest that an ATP-sensitive potassium channel similar to that of mammalian mitochondria is present in A. castellanii mitochondria.

Highlights

These channels can have either detrimental or beneficial effects on the cell
These results suggest that diazoxide stimulates the Kϩ ion flux into A. castellanii mitochondria, decreasing the steady state ⌬⌿ and causing the acceleration of mitochondrial respiration rate
As little is known about monovalent cation-transporting systems in the inner mitochondrial membrane of the unicellular microorganisms and, on the other hand, as potassium channels have been far described only in animal and plant cells, it was considered of utmost importance to study the mitochondrial Kϩ transport mechanism of A. castellanii mitochondria and to compare its properties with those described for other organisms

Summary

EXPERIMENTAL PROCEDURES

Chemicals—L-␣-Phosphatidylcholine (asolectin), diazoxide, glibenclamide, and n-decane were from Sigma-Aldrich. Amoeba A. castellanii SMP (about 5 ␮g of protein/ml, 0.5–1.5 ␮l/reconstitution) were added to the trans compartment (Fig. 4C). The current was measured using a bilayer membrane amplifier (BLM-120, BioLogic). Where Erev is the potential at which the current is zero, R is the gas constant, T is temperature in Kelvin, z is equal to Ϫ1 (chloride anion charge), F is Faraday constant, Pion is the permeability of the ion, and [Cl] and [K] are the respective concentrations of the ion in the cis and trans chambers. Measurements of the respiratory rate were performed in the absence of added ADP, i.e. in the resting state (state 4). High quality mitochondria preparations, i.e. with an ADP/O value of around 1.40 and a respiratory control ratio of around 3 (with succinate), were used in all experiments. Protein bands were visualized using the GE Healthcare ECL system

Membrane by Potassium Channel

DISCUSSION

To further confirm the functional observations obtained