Krzysztof Dołowy

Pharmacology of mitochondrial potassium channels: dark side of the field

Mitochondrial potassium channels play an important role in cytoprotection. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off‐target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. Moreover, understanding their side‐effects should facilitate the design of a specific mitochondrial channel opener with cytoprotective properties. In this review, we discuss the complex interactions of potassium channel inhibitors and activators with cellular structures.

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.

Ion transporting proteins of human bronchial epithelium.

The electrolyte transport system across human airway epithelium followed by water movement is essential for the normal mucociliary clearance that allows the maintenance of the aseptic condition of the respiratory tract. The function of epithelial cells is to control and regulate ionic composition and volume of fluids in the airways. Various types of proteins taking part in assuring effective ions and water transport in apical and basolateral membranes of the airway epithelium have been found (e.g., CFTR, ENaC, CaCC, ORCC, potassium channels, NaKATPase, aquaporins). The paper reviews the current state of the art in the field of ion channels, transporters, and other signaling proteins identified in the human bronchial epithelium. J. Cell. Biochem. 113: 426–432, 2012.

Multielectrode Bisensor System for Time-Resolved Monitoring of Ion Transport Across an Epithelial Cell Layer

An ion-selective multielectrode bisensor system is designed to ensure reliable real-time concentration measurements of sodium, potassium, chloride, and pH in a small volume of biological liquid bathing a living human bronchial epithelial cell monolayer. The bisensor system allows the monitoring of major ions, which are simultaneously transported through the epithelia in both directions.

The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface.

Effect of selected NAD + analogues on mitochondria activity and proliferation of endothelial EA.hy926 cells

The aim of the study was to examine the effect of 1-methylnicotinamide (MNA) and 1-methyl-3-nitropyridine (MNP) on mitochondria activity and proliferation of endothelial EA.hy926 cells. The activity of MNA was also referred to nicotinamide (NAM) being MNA metabolic precursor. NAM and MNA used at high concentrations (up to 1 mM) had no effect on mitochondria metabolism and proliferation of EA.hy926 cells. It could be related to the fact that these compounds hardly cross the cell membrane. It supports the results of our previous study suggesting that anti-inflammatory and anti-thrombotic effects of MNA could be associated with its ability to bind to glycosaminoglycans, especially heparins, located on the endothelium membrane without entering into target cells. In contrast, MNP caused substantial changes in mitochondria activity and proliferation of EA.hy926 cells. This compound used at low concentrations (below 100 microM) blocked the cell cycle of EA.hy926 cells in G1 phase and was very effective in inhibiting cell growth (IC50=13.8+/-2.4 microM). At higher concentrations (0.1-1 mM) MNP caused a significant reduction of cell survival. The observed effects of MNP could be related, at least in part, to its ability to influence the ATP and NAD+ intracellular levels. MNP caused also important changes in Ca2+ intracellular concentration, significant decrease in inner mitochondrial membrane potential and high increase in mitochondrial respiration of EA.hy926 cells. The observed effects of MNP may be related in part to its cellular metabolites detected after 45 min incubation with 250 microM MNP.

POTASSIUM CURRENTS IN HUMAN MYOGENIC CELLS FROM HEALTHY AND CONGENITAL MYOTONIC DYSTROPHY FOETUSES

The whole-cell patch clamp technique was used to record potassium currents in in vitro differentiating myoblasts isolated from healthy and myotonic dystrophy type 1 (DM1) foetuses carrying 2000 CTG repeats. The fusion of the DM1 myoblasts was reduced in comparison to that of the control cells. The dystrophic muscle cells expressed less voltage-activated K+ (delayed rectifier and non-inactivating delayed rectifier) and inward rectifier channels than the age-matched control cells. However, the resting membrane potential was not significantly different between the control and the DM1 cells. After four days in a differentiation medium, the dystrophic cells expressed the fast-inactivating transient outward K+ channels, which were not observed in healthy cells. We suggest that the low level of potassium currents measured in differentiated DM1 cells could be related to their impaired fusion.

Hydrocortisone inhibition of wild-type and αD200Q nicotinic acetylcholine receptors

Short‐term treatment with large doses of corticosteroids can result in acute weakness of muscles in processes that have not yet been fully characterized. Corticosteroids have been shown to exert direct inhibitory action on the muscle‐type nicotinic acetylcholine receptor (AChR), and therefore can promote pharmacological muscle denervation. The mechanism of hydrocortisone (HC) blockage of AChR has not been fully established yet. It is uncommon for an electrically neutral molecule, for example, HC, to induce voltage‐dependent changes in AChR kinetics. Our experiments aimed to determine the source of voltage‐dependency in HC action. Wild‐type (WT) and αD200Q receptors were transiently expressed in HEK293 cells. Recordings were performed in either the presence or absence of HC. We showed that the D‐to‐Q substitution is capable of suppressing the voltage dependency in the HC‐induced block. We conclude that the distance between αD200 and the agonist‐binding site depends on the membrane potential. The voltage‐dependent changes of the αD200 position have not been considered yet. To our knowledge, the ability to induce voltage‐dependency in blocker action has not been shown previously for an amino acid located outside the transmembrane portion of the receptor. Possible mechanisms of HC block (allosteric and knocking) in WT and αD200Q receptors are discussed.

Measurement of ion fluxes across epithelia

Epithelial tissues line all wet surfaces of vertebrate bodies. Their major function is directional transport of ions and water. Cells forming an epithelial layer are bound together by a tight junction that forms a barrier to ion flux. Ions and water are transported via specialized molecules. The presence of a defect in a single ion channel molecule leads to cystic fibrosis - the most common, fatal, human genetic disease. The paper describes ion transport data obtained by means of different experimental techniques. Special attention is given to radiochemical tracers, transepithelial resistance determination, open circuit potential and short circuit current measurements, the nasal potential difference in healthy and cystic fibrosis patients, the use of ion selective electrodes, and electrochemical mapping of the cell membrane surface. The effect of different activators and blockers of ion transport molecules on measured parameters are also discussed.

Multi-electrode system for measurement of transmembrane ion-fluxes through living epithelial cells

Cystic Fibrosis (CF) is the most common fatal human genetic disease. It is caused by the defect in a single anion channel protein which affects ion and water transport across the epithelial tissue. A flat multi-electrode platform of diameter 12mm, allowing for measurement of four ions: sodium, potassium, hydrogen and chloride by exchangeable/replaceable ion-selective electrodes is described. The measurement is possible owing to the architecture of the platform which accommodates all the electrodes and inlets/outlets. The platform fits to the cup and operates in a small volume of the solution bathing the living epithelial cell layer (membrane) deposited on a porous support of the cup, which allows for effective monitoring of ion concentration changes. By applying two multi-electrode platforms, it is possible to measure the ion transmembrane fluxes. The inlet and outlet tubes in the platforms allow for on-fly change of the calibrants, ion-concentration changes and ion channel blockers. Using different ion-concentration gradients and blockers of ion-transporting molecules we show for the first time that sodium ions flow from the basolateral to apical face of the cell monolayer via a paracellular route and return also via a transcellular one, while chloride anions are transported back and forth exclusively via a transcellular route.