Maintenance Of Cell Membrane Potential Research Articles

Metabolic regulation of the two-pore domain potassium channel TASK-1

TASK-1 is a two-pore potassium ion channel and implicated in the maintenance of cell membrane potential. Little is known about the traffic regulation of TASK-1 under metabolic stress. The present study was designed to explore whether cell surface expression of TASK-1 is modulated by hypoxia. The trafficking of recombinant TASK-1 channels was studied in HEK293T cells under hypoxia induced by sodium cyanide (NaCN). Incubation of cells with NaCN significantly reduced TASK-1 fluorescence intensity at cell periphery, and this effect was inhibited by pretreatment with bisindolylmaleimide (BIM), a specific protein kinase C (PKC) inhibitor. Similar results were confirmed by quantifying extracellular HA-tagged TASK-1 on the cell surface using chemiluminescence assay. NaCN-induced reduction in TASK-1 at cell periphery was also prevented by co-expressing the dominant-negative dynamin 1 (K44E), or cholesterol depletion with MβCD, indicating a potential role of cholesterol and dynamin-dependent endocytosis in PKC-mediated reduction of cell surface TASK-1 by NaCN. Mutagenesis studies further showed that NaCN-induced reduction of TASK-1 at cell periphery was observed in cells expressing wild-type TASK-1 but not mutant TASK-1 with mutation of the dileucine motif (263-264 LL-AA) or the PKC phosphorylation consensus site (T400A). Moreover, Western blot analysis demonstrated that NaCN reduced the amount of wild-type TASK-1 but not the TASK-1 mutants (263-264 LL-AA and T400A) in the membrane fraction. In conclusion, our results demonstrate that cell surface TASK-1 channels are downregulated under metabolic inhibition, possibly through PKC-mediated endocytosis in a cholesterol and dynamin-dependent manner. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Different Membrane Environments Generate Multiple Functions of P-type Ion Pumps

P-type ion pumps (P-type ATPases) are involved in various fundamental biological processes. For example, the gastric proton pump (H+,K+-ATPase) and sodium pump (Na+,K+-ATPase) are responsible for secretion of gastric acid and maintenance of cell membrane potential, respectively. In this review, we summarize three topics of our studies. The first topic is gastric H+,K+-ATPase associated with Cl--transporting proteins (Cl-/H+ exchanger ClC-5 and K+-Cl- cotransporter KCC4). In gastric parietal cells, we found that ClC-5 is predominantly expressed in intracellular tubulovesicles and that KCC4 is predominantly expressed in the apical membrane. Gastric acid (HCl) secretion may be accomplished by the two different complexes of H+,K+-ATPase and Cl--transporting protein. The second topic focuses on the Na+,K+-ATPase α1-isoform (α1NaK) associated with the volume-regulated anion channel (VRAC). In the cholesterol-enriched membrane microdomains of human cancer cells, we found that α1NaK has a receptor-like (non-pumping) function and that binding of low concentrations (nM level) of cardiac glycosides to α1NaK activates VRAC and exerts anti-cancer effects without affecting the pumping function of α1NaK. The third topic is the Na+,K+-ATPase α3-isoform (α3NaK) in human cancer cells. We found that α3NaK is abnormally expressed in the intracellular vesicles of attached cancer cells and that the plasma membrane translocation of α3NaK upon cell detachment contributes to the survival of metastatic cancer cells. Our results indicate that multiple functions of P-type ion pumps are generated by different membrane environments and their associated proteins.

Voltage gated proton channels modulate mitochondrial reactive oxygen species production by complex I in renal medullary thick ascending limb

Hv1 is a voltage-gated proton channel highly expressed in immune cells where, it acts to maintain NAD(P)H oxidase activity during the respiratory burst. We have recently reported that Hv1 is expressed in cells of the medullary thick ascending limb (mTAL) of the kidney and is critical to augment reactive oxygen species (ROS) production by this segment. While Hv1 is associated with NOX2 mediated ROS production in immune cells, the source of the Hv1 dependent ROS in mTAL remains unknown. In the current study, the rate of ROS formation was quantified in freshly isolated mTAL using dihydroethidium and ethidium fluorescence. Hv1 dependent ROS production was stimulated by increasing bath osmolality and ammonium chloride (NH4Cl) loading. Loss of either p67phox or NOX4 did not abolish the formation of ROS in mTAL. Hv1 was localized to mitochondria within mTAL, and the mitochondrial superoxide scavenger mitoTEMPOL reduced ROS formation. Rotenone significantly increased ROS formation and decreased mitochondrial membrane potential in mTAL from wild-type rats, while treatment with this inhibitor decreased ROS formation and increased mitochondrial membrane potential in mTAL from Hv1−/− mutant rats. These data indicate that NADPH oxidase is not the primary source of Hv1 dependent ROS production in mTAL. Rather Hv1 localizes to the mitochondria in mTAL and modulates the formation of ROS by complex I. These data provide a potential explanation for the effects of Hv1 on ROS production in cells independent of its contribution to maintenance of cell membrane potential and intracellular pH.

Pterostilbene ameliorates intracerebroventricular streptozotocin induced memory decline in rats.

There is strong evidence that mitochondrial dysfunction mediated oxidative stress results in aging and energy metabolism deficits thus playing a prime role in pathogenesis of Alzheimer's disease, neuronal death and cognitive dysfunction. Evidences accrued in empirical studies suggest the antioxidant, anticancer and anti-inflammatory activities of the phytochemical pterostilbene (PTS). PTS also exhibits favourable pharmacokinetic attributes compared to other stilbenes. Hence, in the present study, we explored the neuroprotective role of PTS in ameliorating the intracerebroventricular administered streptozotocin (STZ) induced memory decline in rats. PTS at doses of 10, 30 and 50mg/kg, was administered orally to STZ administered Sprague-Dawley (SD) rats. The learning and memory tests, Morris water maze test and novel object recognition test were performed which revealed improved cognition on PTS treatment. Further, there was an overall improvement in brain antioxidant parameters like elevated catalase and superoxide dismutase activities, GSH levels, lowered levels of nitrites, lipid peroxides and carbonylated proteins. There was improved cholinergic transmission as evident by decreased acetylcholinesterase activities. The action of ATPases (Na+ K+, Ca2+ and Mg2+) indicating the maintenance of cell membrane potential was also augmented. mRNA expression of battery of genes involved in cellular mitochondrial biogenesis and inflammation showed variations which extrapolate to hike in mitochondrial biogenesis and abated inflammation. The histological findings corroborated the effective role of PTS in countering STZ induced structural aberrations in brain.

Cold acclimation improves chill tolerance in the migratory locust through preservation of ion balance and membrane potential.

Most insects have the ability to alter their cold tolerance in response to temporal temperature fluctuations, and recent studies have shown that insect cold tolerance is closely tied to the ability to maintain transmembrane ion gradients that are important for the maintenance of cell membrane potential (Vm). Several studies have therefore suggested a link between preservation of Vm and cellular survival after cold stress, but none has measured Vm in this context. We tested this hypothesis by acclimating locusts (Locusta migratoria) to high (31°C) and low temperature (11°C) for 4 days before exposing them to cold stress (0°C) for up to 48 h and subsequently measuring ion balance, cell survival, muscle Vm, and whole animal performance. Cold stress caused gradual muscle cell death, which coincided with a loss of ion balance and depolarization of muscle Vm The loss of ion balance and cell polarization were, however, dampened markedly in cold-acclimated locusts such that the development of chill injury was reduced. To further examine the association between cellular injury and Vm we exposed in vitro muscle preparations to cold buffers with low, intermediate, or high [K+]. These experiments revealed that cellular injury during cold exposure occurs when Vm becomes severely depolarized. Interestingly, we found that cellular sensitivity to hypothermic hyperkalaemia was lower in cold-acclimated locusts that were better able to defend Vm whilst exposed to high extracellular [K+]. Together these results demonstrate a mechanism of cold acclimation in locusts that improves survival after cold stress: increased cold tolerance is accomplished by preservation of Vm through maintenance of ion homeostasis and decreased K+ sensitivity.

Disruption of oxidative phosphorylation and synaptic Na+, K+-ATPase activity by pristanic acid in cerebellum of young rats

AimsPeroxisomal biogenesis disorders (PBD) are inherited disorders clinically manifested by neurological symptoms and brain abnormalities, in which the cerebellum is usually involved. Biochemically, patients affected by these neurodegenerative diseases accumulate branched-chain fatty acids, including pristanic acid (Prist) in the brain and other tissues. Main methodsIn the present investigation we studied the in vitro influence of Prist, at doses found in PBD, on oxidative phosphorylation, by measuring the activities of the respiratory chain complexes I–IV and ATP production, as well as on creatine kinase and synaptic Na+, K+-ATPase activities in rat cerebellum. Key findingsPrist significantly decreased complexes I–III (65%), II (40%) and especially II–III (90%) activities, without altering the activities of complex IV of the respiratory chain and creatine kinase. Furthermore, ATP formation and synaptic Na+, K+-ATPase activity were markedly inhibited (80–90%) by Prist. We also observed that this fatty acid altered mitochondrial and synaptic membrane fluidity that may have contributed to its inhibitory effects on the activities of the respiratory chain complexes and Na+, K+-ATPase. SignificanceConsidering the importance of oxidative phosphorylation for mitochondrial homeostasis and of Na+, K+-ATPase for the maintenance of cell membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission in cerebellum. We postulate that these pathomechanisms may contribute to the cerebellar alterations observed in patients affected by PBD in which Prist is accumulated.

Inhibition of Kir2.1 (KCNJ2) by the AMP-activated protein kinase

The inward rectifier K+ channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen–Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKβ1+AMPKγ1), of the constitutively active γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2S795A lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2S795A was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2.

Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) Potassium Channels: VOLATILE ANESTHETICS AND NEUROTRANSMITTERS SHARE A MOLECULAR SITE OF ACTION

TASK-1 and TASK-3, members of the two-pore-domain channel family, are widely expressed leak potassium channels responsible for maintenance of cell membrane potential and input resistance. They are sites of action for a variety of modulatory agents, including volatile anesthetics and neurotransmitters/hormones, the latter acting via mechanisms that have remained elusive. To clarify these mechanisms, we generated mutant channels and found that alterations disrupting anesthetic (halothane) activation of these channels also disrupted transmitter (thyrotropin-releasing hormone, TRH) inhibition and did so to a similar degree. For both TASK-1 and TASK-3, mutations (substitutions with corresponding residues from TREK-1) in a six-residue sequence at the beginning of the cytoplasmic C terminus virtually abolished both anesthetic activation and transmitter inhibition. The only sequence motif identified with a classical signaling mechanism in this region is a potential phosphorylation site; however, mutation of this site failed to disrupt modulation. TASK-1 and TASK-3 differed insofar as a large portion of the C terminus was necessary for the full effects of halothane and TRH on TASK-3 but not on TASK-1. Finally, tandem-linked TASK-1/TASK-3 heterodimeric channels were fully modulated by anesthetic and transmitter, and introduction of the identified mutations either into the TASK-1 or the TASK-3 portion of the channel was sufficient to disrupt both effects. Thus, both anesthetic activation and transmitter inhibition of these channels require a region at the interface between the final transmembrane domain and the cytoplasmic C terminus that has not been associated previously with receptor signal transduction. Our results also indicate a close molecular relationship between these two forms of modulation, one endogenous and the other clinically applied.