K+ influx triggers slow K+/H+ exchange detected by biphasic changes in matrix pH in Guinea pig cardiomyocyte mitochondria
Authors: Lambert David S , Bevers Kyle S , Heisner James S , Camara Amadou KS , Kwok Wai‐Meng , Stowe David F 
Published: June 2021

Recent findings in cell models supports the importance of cation homeostasis by a K+/H+ exchanger (KHE) encoded by the gene LETM1, although LETM1 may also encode Ca2+/H+ exchange and or Na+/H+ exchanger (mNCE). In mitochondria, K+ influx occurs through K+ channels sensitive to Ca2+, Na+, low ATP, and other ligands. K+ influx promotes mitochondrial (m) swelling and contributes to regulation of mitochondrial respiration, membrane potential (mMP) and redox signaling. The influx of mK+ requires a mK+ efflux mechanism (mK+ cycle) paired with H+ influx, which in turn would affect mitochondrial bioenergetics, e.g. respiration, ADP phosphorylation, and mMP due effectively to a “proton leak”. We have previously reported that increased cytosolic and mCa2+ stimulate small and large Ca2+‐sensitive mK+ channels to permit mK+ influx. Cytosolic and mCa2+ increase during ischemia or hypoxia in association with an increase in mitochondrial volume so a high mK+ concentration may be a parallel effect of mCa2+ loading. Experimental conditions that modulate mKHE activity in mammalian cardiac tissue and its consequences have not been examined. Our aim was to provide evidence of mKHE activity in mitochondria isolated from Guinea pig cardiac cell mitochondria and to better understand the circumstances and kinetics of mKHE activity. We proposed that the subsequent efflux of mK+ via mKHE would re‐establish mK+ homeostasis but also provide a “H+ leak” thereby affecting bioenergetics activity. We reasoned that mKHE becomes active once K+ has accumulated in the matrix and that K+ efflux is linked to H+ influx as a marker of mKHE activity. mKHE might be affected by extra‐matrix pH (pHm) as it can change the trans‐membrane pH gradient. To test these ideas, we first added CGP37157 to the mitochondrial buffer to inhibit mNCE. Then we added 25 nM valinomycin, a K+ ionophore, and measured the time‐dependent changes in matrix pHm, using the fluorescent probe BCECF‐AM. We found that across the three extra‐matrix pHs of 6.9, 7.15, and 7.6, matrix pHm transiently increased over approximately 100 s and then transiently decreased over the next 100 s with a slower decline over 20 min. The initial increase in pHm may reflect enhanced proton pumping by respiratory complexes and the secondary decrease in pHm may reflect H+ influx in exchange for K+ efflux, via mKHE. When 500 μM quinine was given before valinomycin to attenuate mKHE, we found that the peak increase in pHm (first phase) occurred later and the subsequent decrease in pHm (second phase) also occurred later by 100 s and was slower to fall over 20 min. The changes to pHm in the presence of quinine provide evidence that the delay in pHm changes were due to block of mKHE by quinine. Interestingly the biphasic effects of valinomycin and quinine on pHm did not appear to be dependent on the extra‐matrix pH values of 6.9, 7.15, 7.6. Additional studies will determine concomitant effects of these protocols on respiration, ADP phosphorylation, mMP, and mitochondrial volume.Support or Funding InformationVA Merit BX‐002539‐01 and NIH T35 HL072483This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.