玻璃 微电极技术及其在 植物胞内测量中的应用

玻璃微电极技术及其在植物胞内测量中的应用

汪 晓 丽

Email : lila77@sina.comTel: 7979645, 7978723

植物生理专题讨论之二

内 容 玻璃微电极技术简介

玻璃微电极及离子选择性玻璃微电极 离子选择性玻璃微电极技术

在研究离子跨膜转运中的应用 在研究离子分室化中的应用 展望

玻璃微电极技术简介

Part 1

Glass Microelectrode(The size of a glass microelectrode tip is less than 1 micrometer )

Microelectrode measuring nitrate in leaf cells

Insertion of microelectrode into plant protoplasts

Diagram of a glass microelectrode measuring the membrane potential of a plant cell

Diagram of a liquid membrane double-barrelled ion-selective microelectrode suitable for intracellular recording

Double-barrelled microelectrode

Diameter of the tip is ＜ 1 micrometer

Inset: end view showing the open ends of each barrel.

Electrode B measuring Em caused by the gradient of K+

Electrode A with the nitrate sensor measuring ele-chemical potential combined with two parts:

i) Em caused by the gradient of K+

ii) Elec-chemical potential caused by the concentration gradient of NO3

-

NO3- potential

= A-B(mV)

= 58 log[NO3-]o/ [NO3

-]i

NO3- ion-selective microelectrode work theory

离子选择性微电极法是测量单个细胞内离子活度的唯一方法。

进行胞内测量时主要的优点： 原位测定，不会对细胞造成伤害； 可同时测定单个细胞的跨膜电势梯度和化学势梯度；

可同时测定多种离子； 与其它胞内测量方法相比，相对比较便宜。

最大的缺点是： 只能测量细胞内单点的离子活度。

拉制玻璃针（ Pulling of micropipettes ） 硅化玻璃管内壁（ Silanizaion of the inside surface ） 灌注离子选择性液膜（ Backfilling with sensor ） 灌注电解质溶液（ Backfilling with salt solution ） 校正（ Calibration ） 测量（ Intracellular measurement ） 再次校正（ Recalibration ）

离子选择性微电极技术Ion-selective microelectrode technique

Pulling a double-barelled micropipettes with a vertical puller

Silanization of the glass inside surface to form a hydrophobic layer

Some examples of sensors for liquid membrane ion-selective microelectrodes

IonSensor molecule(s)

Detection limit

Major interfering ions in plant cells

Ca2+ ETH 129 ETH 1001

10 nM 40 nM

H+, K+, Mg2+ H+, K+, Mg2+

Cl- Mn(III)TPPCa 1-5 mM acetate,HCO3-,SCN-,NO3

-, pH > 7.6

H+ Tridodecylamine ETH 1907

>pH 9 pH 9

K+ K+

K+ Valinomycin 100 µM Ca2+, NH+

Mg2+ ETH 5214 200 µM Ca2+, K+

Na+ ETH 227b ETH 157b

3 mM 2 mM

Ca2+, H+, K+ H+, K+

NH4+ Nonactinb 2 μM K+

NO3- MTDDA.NO3 0.5 mM Cl-, NO2

-, SCN-

A schematic representation showing an ideal ion-selective microelectrode calibration curve.

E = E0±s·log [ ai + ΣKi, j pot(aj)

zi/zj ] (Nicolsky-Einsenman Equation)

s = 2.303RT/ ziF = 58mV (20 , z℃ i=1)

Main characteristics defining an ion-selective electrode: Selectivit

y coefficient, Slope, Detection limit, Response time.

Equipments for intracellular recording

microscope

manipulator

electrometer

computer

An example of calibration and recalibration

Y(mM)=1000{{10^{(x (mV)-p1)/p2}} -p3} (x= A-B)pNO = -log [NO3

- activity] p1 = Eo-offset potential of circuitry

p2 = slope or gradient of calibration p3 = detection limit

Nitrate activity (mmol L-1)

Calibration(mV)

Recalibration(mV)

pNO1 100.0 -24.7 -25.1

pNO2 10.0 24.4 25.6

pNO3 1.0 71.9 72.8

pNO4 0.1 111.3 112.3

pNO5 0.01 118.1 124.9

p1 - 75.76 mV -75.21 mV

p2 - 50.51 mM -50.25 mM

p3 0.0001254 mM 0.00009257 mM

玻璃微电极技术在研究离子跨膜转运中的应用

Part 2

•

•

•

e.g. soilK+ 0.5 mMpH 5-6Ca2+ 1 mM

Membrane transport - key process

•Nutrients accumulation e.g. K+ 100 mM•Regulate cytoplasm e.g. pH 7.2, Ca2+ 100 nM•Remove toxic and waste substances•Export products

Brief introduction of membrane transport systemsBrief introduction of membrane transport systems

Overview of transport proteins in the plasma membrane of plant cells and their proposed transport specifities. Transport through most channel proteins (red) and carrier proteins (orange) is energized by the membrane potential (negative on the inside) and proton gradient generated by the plasma membrane H+-ATPase (blue, middle).

Data refer to experiments with Nitella translucens(Spanswick and Williams,1964)

Ion species Em Ecal Ed Type of uptake

Na+ -138 - 67 - 71 passive

K+ -138 -179 + 41 active

Cl- -138 + 99 -237 active

Em — measured electropotential differences

Ecal — calculated electropotential differences

Ed — driving force, = Em – Ecal

For cations, Ed<0 (Em< Ecal), indicates a passive uptake;

Ed>0 (Em> Ecal), indicates an active uptake.

For anions, Ed >0 (Em> Ecal), indicates an active uptake;

Ed<0 (Em< Ecal), indicates a passive uptake.

Assaying nitrate transporter activity in maize roots by microelectrode measurement

(From: McClure PR, et al. 1990. Plant Physiol)

经载体转运的动力学分析经载体转运的动力学分析

Assaying nitrate

transporter activity

in Arabidopsis mutants

by microelectrode

measurement

（ From: Wang R, et al. 1996. Proc Nart Acad USA)

` NO3-NO3

-

NO3-=1mM

pH = 6.0NO3

- = 5 mMpH = 7.2 = -100 mV

Vacuole

PMCytoplasm Bathing medium

Cell wall

Aminoacids

Xylem

ATP

ADP

H+ H+

2 H+ 2 H+

NO3-

NO3-=10~200mM

pH = 5.5 = -10 mV

Assaying ammonium transporter activity in rice roots by microelectrode measurement

(From: Wang MY, et al. 1994. Plant Physiol)

The effect of cyanide (CN-) on the membrane potential

玻璃微电极技术在研究离子分室化中的应用

Part 3

Triple-barrelled ion-selective microelectrodes

pH barrel allows identification of the cell compartment(cytosol or vacuole)

Triple-barrelled ion-selective micro-electrodes

A A - B = NO3-

B B = EmC

C - B = H+

Cell

Using triple-barrelled K+ and H+ -selective microelectrode to identify the compartmentation of barley root cell

(From: Walker DJ, et al. 1995. Plant Physiol)

结 论 与 展 望

Part 4

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2

3

4

5

6 7

Experimental arrangement for leaf cell measurements

谢　谢　！

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