Hypoxia-induced increase in GABA content is essential for restoration of membrane potential and preventing ROS-induced disturbance to ion homeostasis

ABSTRACT The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes fromSaccharomyces cerevisiae, HAL4/SAT4 andHAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.

Download Full-text

Factors affecting membrane permeability and ionic homeostasis in the cold-submerged frog

Journal of Experimental Biology ◽

10.1242/jeb.203.2.405 ◽

2000 ◽

Vol 203 (2) ◽

pp. 405-414 ◽

Cited By ~ 6

Author(s):

P.H. Donohoe ◽

T.G. West ◽

R.G. Boutilier

Keyword(s):

Skeletal Muscle ◽

Membrane Potential ◽

Membrane Permeability ◽

Rana Temporaria ◽

Ion Homeostasis ◽

Resting Membrane Potential ◽

Tissue Water ◽

Factors Affecting ◽

Ion Gradients ◽

Pump Activity

Frogs (Rana temporaria) were submerged at 3 degrees C in either normoxic (P(O2)=155 mmHg, P(O2)=20 kPa) or hypoxic (P(O2)=60 mmHg; P(O2)=8 kPa) water for up to 16 weeks, and denied air access, to mimic the conditions of an ice-covered pond during the winter. The activity of the skeletal muscle Na(+)/K(+) pump over the first 2 months of hibernation, measured by ouabain-inhibitable (22)Na(+) efflux, was reduced by 30 % during normoxia and by up to 50 % during hypoxia. The reduction in Na(+)/K(+) pump activity was accompanied by reductions in passive (22)Na(+) influx and (86)Rb(+) efflux (effectively K(+) efflux) across the sarcolemma. This may be due to a decreased Na(+) permeability of the sarcolemma and a 75 % reduction in K(+) leak mediated by ATP-sensitive K(+) channels (‘K(ATP)’ channels). The lowered rates of (22)Na(+) and (86)Rb(+) flux are coincident with lowered transmembrane ion gradients for [Na(+)] and [K(+)], which may also lower Na(+)/K(+) pump activity. The dilution of extracellular [Na(+)] and intracellular [K(+)] may be partially explained by increased water retention by the whole animal, although measurements of skeletal muscle fluid compartments using (3)H-labelled inulin suggested that the reduced ion gradients represented a new steady state for skeletal muscle. Conversely, intracellular ion homeostasis within ventricular muscle was maintained at pre-submergence levels, despite a significant increase in tissue water content, with the exception of the hypoxic frogs following 4 months of submergence. Both ventricular muscles and skeletal muscles maintained resting membrane potential at pre-submergence levels throughout the entire period of hibernation. The ability of the skeletal muscle to maintain its resting membrane potential, coincident with decreased Na(+)/K(+) pump activity and lowered membrane permeability, provided evidence of functional channel arrest as an energy-sparing strategy during hibernation in the cold-submerged frog.

Download Full-text

KCNQ1 Potassium Channel Expressed in Human Sperm Is Involved in Sperm Motility, Acrosome Reaction, Protein Tyrosine Phosphorylation, and Ion Homeostasis During Capacitation

Frontiers in Physiology ◽

10.3389/fphys.2021.761910 ◽

2021 ◽

Vol 12 ◽

Author(s):

Tian Gao ◽

Kun Li ◽

Fei Liang ◽

Jianmin Yu ◽

Ajuan Liu ◽

...

Keyword(s):

Membrane Potential ◽

Potassium Channels ◽

Tyrosine Phosphorylation ◽

Sperm Motility ◽

Western Blotting ◽

Acrosome Reaction ◽

Ion Homeostasis ◽

Human Sperm ◽

Protein Tyrosine Phosphorylation ◽

Protein Tyrosine

Potassium channels are involved in membrane hyperpolarization and ion homeostasis regulation during human sperm capacitation. However, the types of potassium channels in human sperm remain controversial. The voltage-gated ion channel KCNQ1 is ubiquitously expressed and regulates key physiological processes in the human body. In the present study, we investigated whether KCNQ1 is expressed in human sperm and what role it might have in sperm function. The expression and localization of KCNQ1 in human sperm were evaluated using Western blotting and indirect immunofluorescence. During capacitation incubation, human sperm were treated with KCNQ1- specific inhibitor chromanol 293B. Sperm motility was analyzed using a computer-assisted sperm analyzer. The acrosome reaction was studied using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin staining. Protein tyrosine phosphorylation levels and localization after capacitation were determined using Western blotting and immunofluorescence. Intracellular K+, Ca2+, Cl−, pH, and membrane potential were analyzed using fluorescent probes. The results demonstrate that KCNQ1 is expressed and localized in the head and tail regions of human sperm. KCNQ1 inhibition reduced sperm motility, acrosome reaction rates, and protein tyrosine phosphorylation but had no effect on hyperactivation. KCNQ1 inhibition also increased intracellular K+, membrane potential, and intracellular Cl−, while decreasing intracellular Ca2+ and pH. In conclusion, the KCNQ1 channel plays a crucial role during human sperm capacitation.

Download Full-text

Intracellular Cl− as a signaling ion that potently regulates Na+/HCO3− transporters

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1415673112 ◽

2015 ◽

Vol 112 (3) ◽

pp. E329-E337 ◽

Cited By ~ 30

Author(s):

Nikolay Shcheynikov ◽

Aran Son ◽

Jeong Hee Hong ◽

Osamu Yamazaki ◽

Ehud Ohana ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Potential ◽

Mammalian Cells ◽

Ion Homeostasis ◽

Transport Processes ◽

Transepithelial Transport ◽

High Affinity ◽

Dependent Site ◽

Receptor Binding Protein

Cl− is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl− (Cl−in) as a signaling ion has not been previously evaluated. Here we report that Cl−in functions as a regulator of cellular Na+ and HCO3− concentrations and transepithelial transport through modulating the activity of several electrogenic Na+-HCO3− transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl−in concentrations highlighting the role of GXXXP motifs in Cl− sensing. Regulation of the ubiquitous Na+-HCO3− co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl−in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl−in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl−in interacting site. NBCe1-A is unaffected by Cl−in between 5 and 140 mM. However, deletion of NBCe1-A residues 29–41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl−in. These findings reveal a cellular Cl−in sensing mechanism that plays an important role in the regulation of Na+ and HCO3− transport, with critical implications for the role of Cl− in cellular ion homeostasis and epithelial fluid and electrolyte secretion.

Download Full-text

Measurement of spontaneous neuronal membrane activity by time lapse confocal microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100141238 ◽

1995 ◽

Vol 53 ◽

pp. 972-973

Author(s):

R H. Selinfreund ◽

A. H. Cornell-Bell

Keyword(s):

Membrane Potential ◽

Confocal Microscopy ◽

Patch Clamp ◽

Electrical Activity ◽

Time Lapse ◽

Neuronal Firing ◽

Neuronal Membrane ◽

Pituitary Cells ◽

Patch Clamp Recording ◽

Spontaneous Electrical Activity

Cellular electrophysiological properties are normally monitored by standard patch clamp techniques . The combination of membrane potential dyes with time-lapse laser confocal microscopy provides a more direct, least destructive rapid method for monitoring changes in neuronal electrical activity. Using membrane potential dyes we found that spontaneous action potential firing can be detected using time-lapse confocal microscopy. Initially, patch clamp recording techniques were used to verify spontaneous electrical activity in GH4\C1 pituitary cells. It was found that serum depleted cells had reduced spontaneous electrical activity. Brief exposure to the serum derived growth factor, IGF-1, reconstituted electrical activity. We have examined the possibility of developing a rapid fluorescent assay to measure neuronal activity using membrane potential dyes. This neuronal regeneration assay has been adapted to run on a confocal microscope. Quantitative fluorescence is then used to measure a compounds ability to regenerate neuronal firing.The membrane potential dye di-8-ANEPPS was selected for these experiments. Di-8- ANEPPS is internalized slowly, has a high signal to noise ratio (40:1), has a linear fluorescent response to change in voltage.

Download Full-text

Fluorescent potentiometric dyes for membrane-potential imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168566 ◽

1994 ◽

Vol 52 ◽

pp. 166-167

Author(s):

Leslie M. Loew

Keyword(s):

Membrane Potential ◽

Single Cells ◽

Quantitative Imaging ◽

Optical Recording ◽

Biological Processes ◽

Major Focus ◽

The Past ◽

Excitable Systems ◽

Major Application ◽

The Impact

A major application of potentiometric dyes has been the multisite optical recording of electrical activity in excitable systems. After being championed by L.B. Cohen and his colleagues for the past 20 years, the impact of this technology is rapidly being felt and is spreading to an increasing number of neuroscience laboratories. A second class of experiments involves using dyes to image membrane potential distributions in single cells by digital imaging microscopy - a major focus of this lab. These studies usually do not require the temporal resolution of multisite optical recording, being primarily focussed on slow cell biological processes, and therefore can achieve much higher spatial resolution. We have developed 2 methods for quantitative imaging of membrane potential. One method uses dual wavelength imaging of membrane-staining dyes and the other uses quantitative 3D imaging of a fluorescent lipophilic cation; the dyes used in each case were synthesized for this purpose in this laboratory.

Download Full-text