Summary
Mitochondrial volume regulation depends on K+ movement across the inner membrane and a mitochondrial Ca2+-dependent K+ channel (mitoKCa) reportedly contributes to mitochondrial K+ uniporter activity. Here we utilize a novel KCa channel activator, NS11021, to examine the role of mitoKCa in regulating mitochondrial function by measuring K+ flux, membrane potential (ΔΨm), light scattering, and respiration in guinea-pig heart mitochondria. K+ uptake and the influence of anions were assessed in mitochondria loaded with the K+ sensor PBFI by adding either the chloride (KCl), acetate (KAc) or phosphate (KH2PO4) salts of K+ to energized mitochondria in a sucrose-based medium. K+ fluxes saturated at ~10mM for each salt, attaining maximal rates of 172±17, 54±2.4 and 33±3.8 nmol K+/min/mg in KCl, KAc or KH2PO4, respectively. NS11021 (50nM) increased the maximal K+ uptake rate by 2.5- fold in the presence of KH2PO4 or KAc, and increased mitochondrial volume, with little effect on ΔΨm. In KCl, NS11021 increased K+ uptake by only 30% and did not increase volume. The effects of NS11021 on K+ uptake were inhibited by the KCa toxins charybdotoxin (200nM) or paxilline (1μM). 50nM NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH2PO4, but not in KCl; however, above 1μM, NS11021 decreased RCR and depolarized ΔΨm. A control compound lacking KCa activator properties did not increase K+ uptake or volume, but had similar nonspecific (toxin insensitive) effects at high concentrations. The results indicate that activating K+ flux through mitoKCa mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨm.