Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.
The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18°C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.
1. Injection of mRNA into Oocytes
2. Preparing the Perfusion System
Illustration of the Automate ValveLink 16 perfusion system. The perfusion system uses pressurized nitrogen to push perfusion solutions out of the solution reservoirs, through the perfusion tubings, and to the perfusion pencil and tip. The flow of each stream of perfusion solution is controlled by one reservoir valve and one electronic valve. In this protocol, one of the reservoirs is not pressurized and functions as a waste collector as well as a pressure-release mechanism. (Picture adapted from vendor’s website http://www.autom8.com)
3. Patch Clamping
4. Representative Results
During the preparation of oocytes for patch clamp, the 5-10 min treatment of stripping solution would detach the vitelline membrane from the plasma membrane, which makes possible stripping the vitelline membrane.
The ideal series resistance of the patch pipette is between 1-1.5 MΩ when filled with pipette solution and submerged in bath solution.
Following is a representative recording of wild-type BK channels on an oocyte membrane patch. On the left top, the square waves indicate the voltage applied to the patch – the second step of voltage increases from 50 mV to 200 mV with increment of 50 mV. Below is the corresponding current traces when the perfusion solution has nominal 0 Ca2+ (free [Ca2+] is about 0.5 nM). The increase of current amplitude indicates that the open probability of BK channels increases with voltage. On the right side, the same patch is perfused with 1.0 μM Ca2+ when applied with the same voltage protocol. The current amplitude increases more under the same voltage, indicating that the open probability increases with [Ca2+].
The oocyte expression system is ideal for electrophysiological characterization of voltage-dependent ion channels due to relatively low background of endogenous channels. Furthermore, since it is a transient expression system, it provides an efficient method of performing mutagenesis study of these channels. However, it should be noted that the oocyte expression system is different from that in mammalian cells, thus there may be differences in post-translational modification and association with different subunits. Furthermore, the lipid concentration may differ between the two cells which may affect the channel’s functional properties.
The perfusion tubings, pencil and tip should be cleaned with DI water every time after experiment to avoid clog. Always use fresh bleach to treat the Ag electrode wire to make sure it is coated with AgCl, otherwise recording will be inaccurate. Adjust the configuration settings for data acquisition so that the test potential is exactly equal to the command potential. It would be helpful to keep the patch pipettes clean so use a lid to protect them from dirt and fire-polish them only before use.
The authors have nothing to disclose.
This work was supported by National Institutes of Health grants R01-HL70393 and R01-NS060706 to J.C. J.C. is a Professor of Biomedical Engineering on the Spencer T. Olin Endowment.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Nanoject II Auto-Nanoliter Injector | Drummond Scientific Company | 3-000-204 | ||
P-97 Flaming/Brown Micropipette Puller | Sutter Instruments | P-97 | ||
Perfusion System and Electronic Controller | AutoMate Scientific, Inc. | ValveLink 16 | Inner diameter of perfusion tip: 100 microns | |
Inverted Microscope | Olympus | CKX31 | ||
Glass Pipettes | VWR International | 53432-921 | ||
Flaming/Brown Micropipette Puller | Sutter Instrument Co. | P-97 | ||
Amplifier | Axon Instruments | AXOPATCH 200B | ||
Computer Interface | INSTRUTECH Corporation | ITC-18 | ||
Headstage | Axon Instruments | CV 203BU |