Methods of Measuring Mitochondrial Potassium Channels: A Critical Assessment

Abstract

In this paper, the techniques used to study the function of mitochondrial potassium channels are critically reviewed. The majority of these techniques have been known for many years as a result of research on plasma membrane ion channels. Hence, in this review, we focus on the critical evaluation of techniques used in the studies of mitochondrial potassium channels, describing their advantages and limitations. Functional analysis of mitochondrial potassium channels in comparison to that of plasmalemmal channels presents additional experimental challenges. The reliability of functional studies of mitochondrial potassium channels is often affected by the need to isolate mitochondria and by functional properties of mitochondria such as respiration, metabolic activity, swelling capacity, or high electrical potential. Three types of techniques are critically evaluated: electrophysiological techniques, potassium flux measurements, and biochemical techniques related to potassium flux measurements. Finally, new possible approaches to the study of the function of mitochondrial potassium channels are presented. We hope that this review will assist researchers in selecting reliable methods for studying, e.g., the effects of drugs on mitochondrial potassium channel function. Additionally, this review should aid in the critical evaluation of the results reported in various articles on mitochondrial potassium channels.

Current Challenges of Mitochondrial Potassium Channel Research

“…The majority of techniques to study mitochondrial K + transport were previously developed to characterize plasma membrane potassium channels. Most likely, measuring the mitochondrial swelling is the only unique technique that can follow the influx of K + into mitochondria ( Pereira and Kowaltowski 2021 ; Walewska et al, 2022b ). Unfortunately, this technique cannot distinguish K + flux via specific mitochondrial potassium channels.…”

“…Unfortunately, this technique cannot distinguish K + flux via specific mitochondrial potassium channels. Moreover, the density of some mitochondrial channels is very low ( Laskowski et al, 2016 ; Pereira and Kowaltowski 2021 ; Walewska et al, 2022b ). Application of pharmacological tools is not helping to use mitochondrial swelling in mitochondrial channel studies.…”

“…direct potassium flux measurements, and iii. biochemical techniques that are indirectly related to potassium movement across the inner mitochondrial membrane ( Walewska et al, 2022b ).…”

“…Key knowledge about the biophysical properties of the potassium channels (such as conductance or selectivity) was obtained with the application of the planar lipid bilayer technique and patch-clamp technique ( Szabo and Zoratti 2014 ; Walewska et al, 2022b ). These techniques are well established and widely used for studies of ion channels from the plasma membrane.…”

“…Direct measurement of K + flux across the inner membrane is also possible by applying of fluorescent probes, i.e., small molecule probes, genetically encoded probes, and thallium (Tl + )-sensitive indicators ( Walewska et al, 2022b ). All these techniques are not easy to apply to study mitochondrial potassium channels in intact cells.…”