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.
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 (Ij) between two opposed Xenopus oocytes.
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 innexins1,2,3. Humans have 21 connexin genes4, while the commonly used invertebrate models C. elegans and Drosophila melanogaster have 25 and 8 innexin genes, respectively5,6. Alternative splicing of gene transcripts may further increase the diversity of GJ proteins, at least for innexins7,8.
GJs may be divided into three categories based on molecular compositions: homotypic, heterotypic, and heteromeric. A homotypic GJ has all its subunits being identical. A heterotypic GJ has two homomeric hemichannels, but the two hemichannels are formed by two different GJ proteins. A heteromeric GJ contains at least one heteromeric hemichannel. The molecular diversities of GJs may confer distinct biophysical properties that are important to their physiological functions. GJ biophysical properties are also modulated by regulatory proteins9. To understand how GJs perform their physiological functions, it is important to know their molecular compositions, biophysical properties, and the roles of regulatory proteins in their functions.
Heterologous expression systems are often used to study biophysical properties of ion channels, including GJs, and the effects of regulatory proteins on them. Because heterologous expression systems allow the expression of specific proteins, they are generally more amenable to dissecting protein functions than native tissues where proteins with redundant functions can complicate the analysis, and recording of Ij can be unattainable. Unfortunately, most commonly used cell lines except the Neuro-2A cell are inappropriate for studying GJ biophysical properties due to complications by endogenous connexins. Even Neuro-2A cells are not always appropriate for this kind of analysis. For example, we could not detect any Ij in Neuro-2A cells transfected with the innexins UNC-7 and UNC-9 in either the absence or the presence of UNC-1 (unpublished), which is required for the function of UNC-9 GJs in C. elegans9,10. On the other hand, Xenopus oocytes are a useful alternative system for electrophysiological analyses of GJs. Although they express an endogenous GJ protein, connexin 38 (Cx38)11, potential complications can be easily avoided by injecting a specific antisense oligonucleotide12. However, analyses of GJs with Xenopus oocytes require a differential voltage clamp of two juxtaposed cells, which is technically challenging. The earliest successes of double voltage clamp of frog blastomeres were reported about 40 years ago13,14. Since then, many studies have used this technique to record Ij in paired Xenopus oocytes. However, essentially all the previous studies have been performed with either homemade amplifiers12,15,16 or commercial amplifiers designed for recordings on single oocytes (GeneClamp 500, AxoClamp 2A, or AxoClamp 2B, Axon Instruments, Union City, CA)8,17,18,19,20. Because even the commercial amplifiers do not provide instructions for double oocyte voltage clamp, it is often challenging for new or less sophisticated electrophysiological labs to implement this technique.
Only one commercial amplifier has been developed for double oocyte voltage clamp, the OC-725C from Warner Instruments (Table of Materials, Figure 1A). This amplifier may be used in either a standard mode (for single oocytes) or a high side current measuring mode (for single or dual oocytes) depending on whether two sockets in its voltage probe are connected (Figure 1B, C). However, until our recent study7, there had not been a single publication describing the use of this amplifier in its high side current measuring mode. Although the amplifier has been used by another lab for dual oocyte recordings, it was used in the standard rather than the high side mode21,22. This lack of reports using the amplifier in its high side current measuring mode might be due to technical difficulties. We were unable to obtain stable dual oocyte recordings using the high side mode by following instructions from the manufacturer. Over the years, we have tried three different approaches for dual oocyte recordings, including using two OC-725C amplifiers in the high side current measuring mode, two OC-725C amplifiers in the standard mode, and two amplifiers from another manufacturer. We eventually succeeded in obtaining stable recordings only with the first approach after extensive trial and error. This publication describes and demonstrates the procedures we use to express GJ proteins in Xenopus oocytes, record Ij using the high side current measuring mode, and analyze the electrophysiological data using popular commercial software. Additional information about the double voltage-clamp technique may be found in other publications19,23.
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
2. GJ protein expression
3. Oocyte pairing
4. Acquisition system preparation
5. Recording Ij between paired oocytes
6. Data analysis
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 terminals7,8. These innexins may form homotypic as well as heterotypic (of UNC-7 and UNC-9) GJs when expressed in Xenopus oocytes7,8. Representative Ij traces and the resulting normalized Gj–Vj relationships of UNC-7b and UNC-9 homotypic GJs are shown in Figure 3. In these experiments with paired oocytes, Vm of Oocyte 1 were clamped to different levels from the holding voltage (-30 mV), whereas that of Oocyte 2 was kept constant at -30 mV to monitor the Ij. The results show that these two types of GJs differ in the Vj-dependent Ij inactivation rate, Vj dependence (indicated by the slope of the Gj–Vj curve), and the amount of the residual Gj. Many other examples of UNC-7 and UNC-9 GJs, including rectifying GJs, may be found in our recent publication7.
Figure 1: Oocyte pairing chamber and amplifier setup. (A) Front panel of the oocyte clamp amplifier OC-725C. (B) A DIP switch inside the amplifier configured for the high side current measurement. All the toggle switches except 2, 5, and 7 are in the OFF position to use the amplifier in the high side current measuring mode. (C) A VDIFF probe with the red socket (for VDIFF input) and black socket (for Circuit Ground) either unconnected or connected. The probe may be used for the standard voltage-clamp mode when the two sockets are connected. (D) An oocyte pairing chamber. (E) A model cell with connections for testing the acquisition system with the amplifier in the high side current measuring mode. Please click here to view a larger version of this figure.
Figure 2: Oocyte and electrode setup. (A) The recording stage. The circular hole has a diameter of 36 mm. (B) Diagram showing the positions of the various electrodes. (C) Actual layout of the various electrodes. (D) Two VDIFF electrodes with their holders and connection cables clamped on the recording stage by two different magnetic clamps, including a modified Agar Bridge Magnetic Holder (Table of Materials) (top) and a tube clamp (Table of Materials) (bottom). The former is more stable in maintaining the electrode position because of its larger magnetic base but requires modification. The glass micropipettes are rotated 90° from their operating positions in order to show the bending angles. (E) A close-up view of a pair of oocytes and the electrodes. Please click here to view a larger version of this figure.
Figure 3: Representative recording traces. (A) Diagram showing the oocyte experiment. Negative and positive membrane voltage (Vm) steps are applied to Oocyte 1 from a holding Vm of -30 mV whereas Oocyte 2 is held at a constant Vm of -30 mV. The transjunctional voltage (Vj) is defined as Vm of Oocyte 2 –Vm of Oocyte 1. (B). Sample Ij traces and the resulting normalized junctional conductance (Gj) –Vj relationship of UNC-9 homotypic gap junctions. (C) Sample Ij traces and the resulting normalized Gj–Vj relationship of UNC-7b homotypic gap junctions. The Gj–Vj relationships are fitted by a Boltzmann function (red lines). Please click here to view a larger version of this figure.
Step No. | Heat | Pull | Velocity | Time |
1 | 260 | … | 40 | 200 |
2 | 240 | … | 40 | 200 |
3 | 240 | 60 | 40 | 200 |
4 | 245 | 100 | 60 | 200 |
Refer to the user manual at the manufacturer’s website for definitions of the pulling parameters (https://www.sutter.com/MICROPIPETTE/p-97.html). |
Table 1: Electrode pulling parameters. These parameters are based on thin-wall glass capillaries (Table of Materials) and a ramp temperature of 258 at a P-97 micropipette puller (Table of Materials). They need to be adjusted according to the ramp temperature for this glass on your puller. For example, if the ramp temperature on the puller is 20° higher, add 20° to each step and make necessary adjustments. Generally, tip size may be optimized by adjusting the velocity of the last step. Please refer to the user manual at the manufacturer's website for meanings of the pulling parameters (https://www.sutter.com/MICROPIPETTE/p-97.html).
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. Second, the VDIFF electrodes must have low resistance (<150 kΩ) and be close to the oocytes. Third, all the electrodes for the same oocyte (voltage, current, and VDIFF) must be positioned on the same side (left or right), and the order of the electrodes (from back to front) should be current, voltage, and VDIFF. Lastly, the reference electrode should be located near the edge of the 35-mm Petri dish toward the user.
We have modified a few recommended procedures from the manufacturer and made some other improvements. Among them are: 1) ND96-filled micropipettes instead of KCl-loaded agar bridges to serve as VDIFF electrodes. The glass electrodes are easy to construct, reusable, and non-harmful to oocytes; 2) a low leakage KCl electrode as the reference electrode. This electrode has low resistance (~2.7 kΩ) and stable potential, and leaks little electrolytes (~5.7 x 10-8 mL/h); 3) a custom-designed and constructed recording platform that allows stable, convenient, and precise positioning of oocytes and the various electrodes. This stage also provides ample access to a stereomicroscope, a fiber light with dual goosenecks, and the four magnetic stands used to mount and position the current and voltage electrodes; and 4) fabrication of current and voltage electrodes from a type of thin-wall glass capillaries. These electrodes have the desired tip resistance (0.5-1.4 MΩ), can penetrate the oocyte cell membrane very easily, and cause minimum damage to oocytes.
Occasionally, the recording system does not work properly, as indicated by an unusually large or continuously increasing holding current, development of a white spot in the cell membrane around the current electrode, and unstable Vm traces in response to voltage commands. The possible causes are 1) a VDIFF electrode system has a small air bubble or the tip of the VDIFF electrode is not aimed properly toward the oocyte; 2) a voltage or current electrode has high resistance (e. g. >2 MΩ) or its holder is dirty from salt deposit; 3) the connection wire for a VDIFF electrode is broken; 4) the D.C. gain is not set to IN during voltage clamp.
Here we have described a method for recording Ij from Xenopus oocytes. It allows a stable voltage clamp of two opposed oocytes. This method is easy to implement and appears to have no obvious limitations for analyzing the biophysical properties of GJs. However, we are not in a position to tell how it compares with other published methods. We hope that labs that are newly interested in setting up the double oocyte voltage-clamp technique will find this method worth considering.
The authors have nothing to disclose.
We thank Haiying Zhan, Qian Ge for their involvement in the initial stage of technical development, Kiranmayi Vedantham for helping with the figures, and Dr. Camillo Peracchia for advice on the oocyte pairing chamber.
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 |