ना, कश्मीर और एच, कश्मीर ATPase में कटियन परिवहन मापने<em> Xenopus</em> परमाणु अवशोषण स्पेक्ट्रोफोटोमेट्री द्वारा oocytes: रेडियोआइसोटोप assays के लिए एक वैकल्पिक

Summary

We describe a method to quantify the activity of K⁺-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb⁺ or Li⁺ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Abstract

Whereas cation transport by the electrogenic membrane transporter Na⁺,K⁺-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H⁺,K⁺-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb⁺ or Li⁺ transport by Na⁺,K⁺– or gastric H⁺,K⁺-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb⁺ (Li⁺) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb⁺ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na⁺/2K⁺ transport stoichiometry of the Na⁺,K⁺-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H⁺,K⁺-ATPase. In principle, the assay is not limited to K⁺-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Introduction

We wanted to develop a sensitive, safe and inexpensive alternative to radioactive tracer experiments to investigate the specific transport activity of ion translocating membrane proteins in order to circumvent restrictions regarding the access to isotope laboratories, safety requirements or the use of costly radioisotopes, which – as in the case of lithium – may even be unavailable due to extremely short decay times. We were particularly interested in determining the activity of the electroneutrally operating gastric H⁺,K⁺-ATPase, because the enzyme does not generate current and its activity can therefore not be addressed by electrophysiological methods. Since Na⁺,K⁺– and H⁺,K⁺-ATPase transport Rb⁺ as efficient as K⁺ (and Li⁺ as well), the high sensitivity of the AAS technique for rubidium or lithium should facilitate sensitive detection of transport activity. Atomic absorption spectrophotometers are common analytical devices, which are widely distributed in chemical laboratories and should be accessible to a large number of interested scientists. Furthermore, we wanted to take advantage of the Xenopus oocyte expression system, which utilizes large single cells (about 1.0-1.5 mm diameter) that allow to achieve a remarkably low cell-to-cell variability regarding the protein expression level within a single batch. A simple calculation demonstrates the feasibility of the AAS assay: The detection limit (characteristic mass) for rubidium with the THGA-AAS technique is 10 pg or 1.2·10^-13 mol (Rb: 85.47 g/mol), for lithium 5.5 pg or 7.9·10^-13 mol (Li: 6.94 g/mol). Upon heterologous expression of Na⁺,K⁺-ATPase in Xenopus oocytes, pump currents of 100 nA can be achieved (which equals about 6.2·1011 elementary charges per second, or 1.03·10^-12 mol s^-1), thus resulting in a transport of 6·10^-6 C of charge within 1 min. Since the transport of one net charge corresponds to the uptake of two Rb⁺ ions (due to the 3Na⁺/2K⁺ stoichiometry), 100 nA current for 1 min corresponds to an uptake of 1.2·10^-10 mol Rb⁺. Thus, even upon a 1,000-fold dilution (homogenization of an oocyte with about 1 μl volume in 1 ml water), a typical THGA-AAS sample (20 μl) contains 2.4·10^-12 mol Rb⁺ (or 204 pg), which is far above the detection threshold. Therefore, even transporters with more than 100-fold lower transport activity or plasma membrane expression can be assayed with the technique by appropriately adjusting the flux time of the experiment.

Since the pumping rate is sensitively dependent on temperature (typical activation energies for Na⁺,K⁺-ATPase are in the range of 90 kJ/mol to 130 kJ/mol^1-3, which results in an about 30% increase in the turnover rate upon a change from 20 °C to 22 °C), it is mandatory to carry out the flux measurements under precise temperature control (air conditioning) with well equilibrated buffer solutions. Furthermore, oocytes should be carefully selected regarding homogenous size for the expression of an ion transporter. With these precautions, it is possible to routinely achieve experimental standard errors of less than 10 percent with about 10 cells per experimental condition. Using this technique, we were able to determine e.g. the apparent Rb⁺ affinities of cation transport^4-6, the influence of extra- and intracellular pH⁷ and the effect of mutations of residues involved in cation coordination during transport^4,8. An advantage of the technique is that ion fluxes can also be determined in combination with two-electrode voltage clamping of the oocytes, which on one hand assures accurate control of the membrane potential during transport and on the other hand allows to correlate ion flux with membrane current. Thus, it was possible to verify the 3Na⁺/2K⁺ stoichiometry of the Na⁺,K⁺-ATPase (see exemplary results below) and to determine the voltage dependence of cation transport of the gastric H⁺/K⁺-ATPase⁷.

Protocol

1. cDNA Constructs and Protein Expression in Xenopus Oocytes The cDNA of the membrane protein of interest should be cloned into a vector suitable for expression in Xenopus laevis oocytes such as pTLN9 or pcDNA3.1X10. Such optimized vectors contain the 5′- and 3′-untranslated regions (UTR) of the Xenopus laevis β-globin gene flanking the multiple cloning site (MCS), an RNA polymerase promoter sequence (pTLN: SP6, pcDNA3.1X: T7) located before …

Representative Results

Quantification of Rb+ uptake by K+- (or Rb+)-countertransporting P-type ATPases by AAS in the Xenopus oocyte expression system permits reliable determination of enzyme kinetic parameters. Determination of the transport stoichiometry of the Na+/K+-ATPase For the electrogenic Na+/K+-ATPase, Rb+ fluxes can be determined in two-electrode voltage-clamp experiments aimed at the c…

Discussion

The described method to measure the amount of Rb⁺ (or Li⁺) taken up into individual Xenopus oocytes expressing Na⁺,K⁺– or H⁺,K⁺-ATPase has proven to be a versatile, flexible and accurate technique to determine the kinetic or thermodynamic parameters of transport for cation-countertransporting P-type ATPases^1,4,5,7,8. It is a safe and reliable alternative to radioactive tracer flux assays, and allows addressing a large scope of experimen…

Materials

Name of the Reagent	Company	Catalogue Number	Comments (optional)
4.0 Ethicon Vicryl suture material	Johnson & Johnson	V633H
Collagenase type 1A from Clostridium hystolyticum	Sigma Aldrich	C9891
High-Pure PCR Product Purification Kit	Roche Applied Science	11732676001
Nuclease-free water	Ambion	AM9937
mMessage mMachine Kit SP6/T7	Ambion	1340, 1344
Ouabain octahydrate	Sigma Aldrich	O3125
Tricain (ethyl 3-aminobenzoate methanesulfonate salt)	Sigma Aldrich	A5040
Trypsin inhibitor type III-O from chicken egg white	Sigma Aldrich	T2011
SCH28080	Sigma Aldrich	S4443
AAnalyst 800	Perkin Elmer	0993-5256
WinLab32TM	Perkin Elmer
BioPhotometer	Eppendorf	6131 000.012
Borosilicate Capillaries	Science Products	GB150F-8P
Hematocrit tubes 3.5″	Drummond Scientific	3 000-203-G/X
Hollow Cathode Lamp Lithium	Photron	P929LL
Hollow Cathode Lamp Rubidium	Photron	P945
Micropipette Puller	Narishige	Model PC-10
Oocyte Recording Chamber RC-10	Warner Instr.	W4 64-0306
Nanoject II Injection Pump	Drummond Scientific	3-000-204
pCLAMP software	Molecular Devices
Polypropylene Sample Cup (1.2 ml)	Perkin Elmer	B0510397
Speedvac – Concentrator model 5301	Eppendorf	5301 000.210
THGA Tube	Perkin Elmer	B3000641
Turbo TEC-10CX Amplifier	NPI Electronics	TEC-10CX