scholarly journals Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels

2019 ◽  
Vol 20 (20) ◽  
pp. 4995 ◽  
Author(s):  
León-Aparicio ◽  
Salvador ◽  
Aparicio-Trejo ◽  
Briones-Herrera ◽  
Pedraza-Chaverri ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Rat Kidney ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Immunogold Electron Microscopy ◽  
Hcn Channels ◽  
Inner Mitochondrial Membrane ◽  
Action Potential Firing ◽  
Human Embryonic Kidney Cells ◽  
Kidney Mitochondria

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1–4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.

scholarly journals Conditions for activity of glutaminase in kidney mitochondria

1970 ◽  
Vol 118 (2) ◽  
pp. 265-274 ◽  
Author(s):  
Z. Kovačević ◽  
J. D. McGivan ◽  
J. B. Chappell
Keyword(s):  
Enzyme Activity ◽  
Mitochondrial Membrane ◽  
Energy State ◽  
Rat Kidney ◽  
Antimycin A ◽  
Ph Optimum ◽  
Rapid Reduction ◽  
Kidney Mitochondria ◽  
Glutaminase Activity

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)+ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, Pi and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with Pi. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K+-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with Pi and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H+ ions, which together with accumulation of Pi increases glutaminase activity. Translocation of K+ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of Pi. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.

Cd2+-induced swelling-contraction dynamics in isolated kidney cortex mitochondria: role of Ca2+ uniporter, K+ cycling, and protonmotive force

2005 ◽  
Vol 289 (3) ◽  
pp. C656-C664 ◽  
Author(s):  
Wing-Kee Lee ◽  
Malte Spielmann ◽  
Ulrich Bork ◽  
Frank Thévenod
Keyword(s):  
Mitochondrial Membrane ◽  
Rat Kidney ◽  
Choline Chloride ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Kidney Cortex ◽  
Protonmotive Force ◽  
Inner Mitochondrial Membrane ◽  
Rat Kidney Cortex ◽  
The Matrix

The nephrotoxic metal Cd2+ causes mitochondrial damage and apoptosis of kidney proximal tubule cells. A K+ cycle involving a K+ uniporter and a K+/H+ exchanger in the inner mitochondrial membrane (IMM) is thought to contribute to the maintenance of the structural and functional integrity of mitochondria. In the present study, we have investigated the effect of Cd2+ on K+ cycling in rat kidney cortex mitochondria. Cd2+ (EC50 ∼19 μM) induced swelling of nonenergized mitochondria suspended in isotonic salt solutions according to the sequence KCl = NaCl > LiCl ≫ choline chloride. Cd2+-induced swelling of energized mitochondria had a similar EC50 value and showed the same cation dependence but was followed by a spontaneous contraction. Mitochondrial Ca2+ uniporter (MCU) blockers, but not permeability transition pore inhibitors, abolished swelling, suggesting the need for Cd2+ influx through the MCU for swelling to occur. Complete loss of mitochondrial membrane potential (ΔΨm) induced by K+ influx did not prevent contraction, but addition of the K+/H+ exchanger blocker, quinine (1 mM), or the electroneutral protonophore nigericin (0.4 μM), abolished contraction, suggesting the mitochondrial pH gradient (ΔpHm) driving contraction. Accordingly, a quinine-sensitive partial dissipation of ΔpHm was coincident with the swelling-contraction phase. The data indicate that Cd2+ enters the matrix through the MCU to activate a K+ cycle. Initial K+ load via a Cd2+-activated K+ uniporter in the IMM causes osmotic swelling and breakdown of ΔΨm and triggers quinine-sensitive K+/H+ exchange and contraction. Thus Cd2+-induced activation of a K+ cycle contributes to the dissipation of the mitochondrial protonmotive force.

scholarly journals Use Dependent Attenuation of Rat HCN1-Mediated Ih in Intact HEK293 Cells

2016 ◽  
Vol 38 (6) ◽  
pp. 2079-2093 ◽  
Author(s):  
Lennart Barthel ◽  
Olivia Reetz ◽  
Ulf Strauss
Keyword(s):  
Mammalian Cells ◽  
Hek293 Cells ◽  
Hcn Channels ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Whole Cell ◽  
Human Embryonic Kidney Cells ◽  
Intracellular Content ◽  
Voltage Dependent ◽  
Activity Dependence

Background/Aims: Cationic currents (Ih) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. Methods: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated Ih in cell-attached or whole-cell mode of the patch-clamp technique. Results: Recurring activation of rHCN1 reduced and slowed Ih in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced Ih attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in Ih. Conclusion: Reduction of rHCN1 mediated Ih is use dependent and may involve the coupling of voltage sensor and pore.

scholarly journals SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential

2021 ◽  
Author(s):  
Nadezda A. Brazhe ◽  
Evelina I. Nikelshparg ◽  
Adil A. Baizhumanov ◽  
Vera G. Grivennikova ◽  
Anna A. Semenova ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Conformational Changes ◽  
Mitochondrial Membrane ◽  
Atp Synthesis ◽  
Surface Enhanced Raman Spectroscopy ◽  
Complex Iii ◽  
Label Free ◽  
Inner Mitochondrial Membrane ◽  
The Impact

AbstractCytochrome c is an essential component of the electron transport chain (ETC), which regulates respiratory chain activity, oxygen consumption, and ATP synthesis. But the impact of conformational changes in cytochrome c on its function is not understood for lack of access to these changes in intact mitochondria. Here we describe a label-free tool that identifies conformational changes in cytochrome c heme and elucidates their function. We verify that molecule bond vibrations assessed by surface-enhanced Raman spectroscopy (SERS) is a reliable indicator of the planar heme configuration during activation of ETC and decrease in inner mitochondrial membrane potential. The planar conformation of cytochrome c heme enables its optimal orientation towards the heme of cytochrome c1 in complex III. This ensures a faster electron transfer, which is important during ETC speed-ups and acceleration of ATP synthesis. The ability of our tool to track mitochondrial function opens wide perspectives on cell bioenergetics.

scholarly journals The Mitochondrial Permeability Transition in Mitochondrial Disorders

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Justina Šileikytė ◽  
Michael Forte
Keyword(s):  
Mitochondrial Membrane ◽  
Mitochondrial Permeability Transition ◽  
Physiological Role ◽  
Mitochondrial Diseases ◽  
Atp Synthesis ◽  
Permeability Transition ◽  
Mitochondrial Disorders ◽  
Molecular Entity ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Permeability

Mitochondrial permeability transition pore (PTP), a (patho)physiological phenomenon discovered over 40 years ago, is still not completely understood. PTP activation results in a formation of a nonspecific channel within the inner mitochondrial membrane with an exclusion size of 1.5 kDa. PTP openings can be transient and are thought to serve a physiological role to allow quick Ca2+ release and/or metabolite exchange between mitochondrial matrix and cytosol or long-lasting openings that are associated with pathological conditions. While matrix Ca2+ and oxidative stress are crucial in its activation, the consequence of prolonged PTP opening is dissipation of the inner mitochondrial membrane potential, cessation of ATP synthesis, bioenergetic crisis, and cell death—a primary characteristic of mitochondrial disorders. PTP involvement in mitochondrial and cellular demise in a variety of disease paradigms has been long appreciated, yet the exact molecular entity of the PTP and the development of potent and specific PTP inhibitors remain areas of active investigation. In this review, we will (i) summarize recent advances made in elucidating the molecular nature of the PTP focusing on evidence pointing to mitochondrial FoF1-ATP synthase, (ii) summarize studies aimed at discovering novel PTP inhibitors, and (iii) review data supporting compromised PTP activity in specific mitochondrial diseases.

Modulation of the calcium-activated BK-channel of the inner mitochondrial membrane by hypoxia

2007 ◽  
Vol 34 (S 2) ◽  
Author(s):  
D Siemen ◽  
Y Cheng ◽  
X Gu ◽  
P Bednarczyk ◽  
GG Haddad ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Bk Channel ◽  
Inner Mitochondrial Membrane
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