Two-electrode voltage clamp of Xenopus oocytes under high hydrostatic pressure

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

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Reversible and irreversible interactions of DIDS with the human electrogenic Na/HCO3 cotransporter NBCe1-A: role of lysines in the KKMIK motif of TM5

AJP Cell Physiology ◽

10.1152/ajpcell.00267.2006 ◽

2007 ◽

Vol 292 (5) ◽

pp. C1787-C1798 ◽

Cited By ~ 41

Author(s):

Jing Lu ◽

Walter F. Boron

Keyword(s):

Voltage Clamp ◽

Dose Response ◽

Response Curve ◽

Xenopus Oocytes ◽

Transmembrane Segment ◽

Initial Value ◽

Irreversible Inhibition ◽

Electrode Voltage

Others have shown that H2DIDS reversibly and covalently binds to the first lysine (K) in the SKLIK motif at the extracellular end of transmembrane segment 5 of the Cl-HCO3 exchanger AE1. Here we mutated K558, K559, and/or K562 in the homologous KKMIK motif of human NBCe1-A. We expressed constructs in Xenopus oocytes, and used a two-electrode voltage clamp to test the sensitivity of the NBC current (−160 to +20 mV) to DIDS. A 30-s DIDS exposure decreased the current at 0 mV, and a subsequent albumin wash returned the current to the initial value (less any irreversible DIDS inhibition), permitting the determination of a complete dose-response curve on a single oocyte. For all constructs, the reversible DIDS inhibition of the NBC current decreased at more negative voltages. The apparent inhibitory constant for reversible DIDS binding increased in the sequence RRMIR < KKMIK ( wt, ∼40 μM) < NKMIK ≅ NKMIN ≅ KKMIN < KNMIN ≅ KNMIK < NNMIK < NNMIN (∼400 μM) < DDMID < EEMIE (∼800 μM). Thus the second K is the most important for reversible DIDS blockade. Nevertheless, these mutations had relatively little effect on slope conductance in the absence of DIDS. For KKMIK, RRMIR, NKMIK, KKMIN, KNMIK, and NNMIN, the rates of irreversible inhibition by DIDS roughly parallel the apparent affinities for reversible DIDS binding. The rate was extremely low for DDMID. The fitted maximal inhibitions were 80–91% for the first five constructs, and 66% for NNMIN. Thus DIDS probably reversibly binds before irreversibly reacting with NBCe1-A. Finally, tenidap blocks not only KKMIK, but also NNMIN and EEMIE.

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Functional Characterization of Connexin Hemichannels Using Xenopus Oocytes and the Two-Electrode Voltage Clamp Technique

Gap Junction Channels and Hemichannels ◽

10.1201/9781315369396-9 ◽

2016 ◽

pp. 203-214 ◽

Cited By ~ 1

Author(s):

Juan Manuel Valdez Capuccino ◽

Jorge E. Contreras

Keyword(s):

Voltage Clamp ◽

Xenopus Oocytes ◽

Functional Characterization ◽

Voltage Clamp Technique ◽

Clamp Technique ◽

Electrode Voltage ◽

Connexin Hemichannels

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Fluctuations in Xenopus oocytes protein phosphorylation levels during two-electrode voltage clamp measurements

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2005.10.005 ◽

2006 ◽

Vol 153 (1) ◽

pp. 62-70 ◽

Cited By ~ 10

Author(s):

Asi Cohen ◽

Noam Zilberberg

Keyword(s):

Protein Phosphorylation ◽

Voltage Clamp ◽

Xenopus Oocytes ◽

Electrode Voltage

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Survival of mouse blastocysts after low-temperature preservation under high pressure

Acta Veterinaria Hungarica ◽

10.1556/avet.52.2004.4.10 ◽

2004 ◽

Vol 52 (4) ◽

pp. 479-487 ◽

Cited By ~ 7

Author(s):

Cs. Pribenszky ◽

M. Molnár ◽

S. Cseh ◽

L. Solti

Keyword(s):

Phase Transition ◽

Hydrostatic Pressure ◽

Low Temperature ◽

High Hydrostatic Pressure ◽

Room Temperature ◽

Freezing Point ◽

Significant Loss ◽

Embryo Cryopreservation ◽

Subzero Temperatures ◽

Survival Capacity

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21°C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0°C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.

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