Recording Gap Junction Current from Xenopus Oocytes

Summary

Here we present a protocol to express gap junction proteins in Xenopus oocytes and record junctional current between two apposed oocytes using a commercial amplifier designed for dual oocyte voltage-clamp recordings in a high side current measuring mode.

Abstract

Heterologous expression of connexins and innexins in Xenopus oocytes is a powerful approach for studying the biophysical properties of gap junctions (GJs). However, this approach is technically challenging because it requires a differential voltage clamp of two opposed oocytes sharing a common ground. Although a small number of labs have succeeded in performing this technique, essentially all of them have used either homemade amplifiers or commercial amplifiers that were designed for single-oocyte recordings. It is often challenging for other labs to implement this technique. Although a high side current measuring mode has been incorporated into a commercial amplifier for dual oocyte voltage-clamp recordings, there had been no report for its application until our recent study. We have made the high side current measuring approach more practical and convenient by introducing several technical modifications, including the construction of a magnetically based recording platform that allows precise placement of oocytes and various electrodes, use of the bath solution as a conductor in voltage differential electrodes, adoption of a commercial low-leakage KCl electrode as the reference electrode, fabrication of current and voltage electrodes from thin-wall glass capillaries, and positioning of all the electrodes using magnetically based devices. The method described here allows convenient and robust recordings of junctional current (I_j) between two opposed Xenopus oocytes.

Introduction

GJs are intercellular channels that may allow the current flow and exchange of small cytosolic molecules between neighboring cells. They exist in many cell types and perform diverse physiological functions. GJs in vertebrates are formed by connexins, whereas those in invertebrates by innexins. Each GJ consists of two juxtaposed hemichannels with either 6 or 8 subunits per hemichannel, depending on whether they are connexins or innexins^1,2,3. Humans have 21 connexin genes⁴, while the commonly used invertebrate models C. elegans and Drosophi....

Protocol

The surgeries are performed following a protocol approved by the institutional animal care committee of the University of Connecticut School of Medicine.

1. Frog surgery and preparation of defollicuated oocytes

1. Anesthetize an adult female African clawed frog (Xenopus laevis) (Table of Materials) by immersing in a cool (with ice) tricaine solution (~300 mg/L).
2. Wait (~15 min) until the frog shows little or no response to squeezing its webbed feet. Lay the frog on an operating table with its belly facing up.
3. After a longitudinal incision (8-10 mm long) on either the lef....

Results

UNC-7 and UNC-9 are innexins of C. elegans. While UNC-9 has only one isoform, UNC-7 has multiple isoforms that differ mainly in the length and amino acid sequence of their amino terminals^7,8. These innexins may form homotypic as well as heterotypic (of UNC-7 and UNC-9) GJs when expressed in Xenopus oocytes^7,8. Representative I_j traces and the resulting normalized .......

Discussion

System optimization appears to be necessary for dual oocyte voltage-clamp experiments. Without it, recordings can be highly unstable, and the amplifiers may have to inject an excessive amount of current to reach the target Vm, resulting in oocyte damage and recording failures. Several factors are critical to obtaining stable dual oocyte recordings with the high side current measuring method. First, the current and voltage electrodes must have appropriate resistance (~1 MΩ), and their holders must be clean........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar Bridge Magnetic Holder	ALA Scientific Instruments	MPSALT-H	More stable than the Narishige tube clamper due to its larger magnetic base but it requires modification to accmmodate a 2-mm female socket.
Auto Nanoliter Injector	Drummond Scientific Company, Broomall, PA, USA	Nanoject II	Automated nanoliter injector
Collagenase, Type II	Gibco-USA, Langley, OK, USA	17101-015
Diamond Scriber	Electron Microscopy Sciences, Hatfield, PA, USA	62108-ST
Differential Voltage Probe	Warner Instruments, Hamden, CT, USA	7255DI
Analog-to-Digital Signal Converter	Molecular Devices, San Jose,CA, USA	Digidata 1440A
Dumont #5 Tweezers	World Precision Instruments, Sarasota, FL, USA	500341
Glass Capillaries	Drummond Scientific Company, Broomall, PA, USA	3-000-203-G/X
Hot Wire Cutter	Amazon.com	Proxxon 37080	An alternative is Hercules 8500 DHWT, which has a foot control pedal.
Hyaluronidase, Type I-S	MilliporeSigma, Burlington, MA, USA	H3506
Magnetic Holder Base	Kanetec USA Corp. , Bensenville, IL, USA	MB-L-45
Microelectrode Beveler	Sutter Instrument, Novato, CA, USA	BV-10
Microelectrode Holder	World Precision Instruments, , Sarasota, FL, USA	MEH1S15
Micropipette Puller	Sutter Instrument, , Novato, CA, USA	P-97
mMESSAGE mMACHINETM T3	Invitrogen-FisherScientific	AM1348
Nunc MicroWell MiniTray	Nalge Nunc International, Rochester, NY, USA	438733	Microwell Minitray
Nylon mesh	Component Supply Company, Sparta, TN, USA	U-CMN-1000
Oocyte Clamp Amplifier	Warner Instruments, , Hamden, CT, USA	OC-725C
OriginPro	OriginLab Corporation, Northampton, MA, USA	2020b
pClamp	Molecular Devices, , San Jose,CA, USA	Version 10
Reference Electrode	World Precision Instruments, Sarasota, FL, USA	DRIREF-2SH	Specifications: https://www.wpiinc.com/blog/post/compare-dri-ref-reference-electrodes
RNaseOUT (ribonuclease inhibitor)	Invitrogen-FisherScientific	10777-019
Silk Suture 5-0	Covidien, North Haven, CT, USA	VS890
Spectrophotometer NanoDrop Lite	Thermo Scientific	ND-LITE-PR
Thin Wall Glass Capallaries	World Precision Instruments,Sarasota, FL, USA	TW150F-4
Tube Clamper	Narishige International USA, Amityville, NY, USA	CAT-1	Ready to use but its position is prone to shift due to the small magnetic base.
Xenopus laevis	Xenopus Express, Brooksville, FL, USA	IMP-XL-FM