Calculation of ion currents across the inner membrane of functionally intact mitochondria

Abstract

Mitochondrial ion transport systems play a central role in cell physiology. Rates of Ca (2+) and K(+) transport across the inner mitochondrial membrane have been derived from the measurement of ion accumulation over time within functional isolated mitochondria or mitochondria of cultured cells. Alternatively, the electrical currents generated by ionic flux have been directly measured in purified and swollen mitochondrial samples (mitoplasts) or reconstituted channels, and typically range from 1 pA to several 100s pA. However, the direct electrophysiological approach necessarily requires extensive processing of the mitochondria prior to measurement, which can only be performed on isolated mitoplasts. To compare rates of mitochondrial ion transport measured in electrophysiological experiments to those measured in intact mitochondria and cells, we converted published rates of mitochondrial ion uptake into units of ionic current. We estimate that for monovalent ions, uptake by intact mitochondria at the rate of 1 nmol ∙ mg(-1) protein ∙ min(-1) is equivalent to 0.2 fA of current per whole single mitochondrion (0.4 fA for divalent ions). In intact mitochondria, estimated rates of electrogenic cation uptake are limited to 1-100 fA of integral current per single mitochondrion. These estimates are orders of magnitude lower than the currents through mitochondrial channels directly measured via patch-clamp or artificial lipid bilayer approaches.

Keywords: calcium; ion transport; lipid bilayer; mitochondria; patch-clamp; potassium.

Figure 1. General overview of the 2 methods used to quantify mitochondrial ion transport, compared in the present report. Both methods begin with rupturing the intact cell to obtain mitochondria. (A) The method using intact mitochondria. Determining mitochondrial ion uptake involves isolating intact mitochondria and measuring the total amount of ions transported into the organelle after a given amount of time. In intact mitochondria, the electrogenic uptake of ions is dependent upon the driving force of the mitochondrial membrane potential (ΔΨ), which is established by the activity of the respiratory chain. (B) The direct method of measuring mitochondrial ion flux across the inner mitochondrial membrane. This method involves first swelling, and rupturing the outer mitochondrial membrane. The patch pipette can either be attached to the resulting mitoplast, or a portion of the mitoplast can be excised for direct measures of ionic currents generated by ionic flux through channel of interest. It is important to highlight that the voltage applied via the patch pipette is analogous to the ΔΨ in intact mitochondria, in that it provides the driving force for ionic uptake.