Name: patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes
Uploaded: 2011-01-10T18:10:00
Description: Watch this Scientific Journal Video about Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes at JoVE.com

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.

1. Injection of mRNA into Oocytes
<ol>
	<li>Inject 0.05 - 50 ng messenger RNA that was transcribed in vitro into Xenopus laevis oocytes (stage V-VI) using Nanoject II Auto-Nanoliter Injector (Drummond Scientific Company, model 3-000-204). Repeat the injection on a dozen oocytes for each mRNA.</li>
	<li>Rinse the injected oocytes twice using ND-96 solution (96 mM sodium chloride (NaCl, Mr 58.44 g/mol), 2 mM potassium chloride (KCl, Mr 74.56 g/mol), 1.8 mM calcium chloride (CaCl2&#8226;2H<...

<ol>
	<li>Methfessel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+measurements+on+Xenopus+laevis+oocytes:+currents+through+endogenous+channels+and+implanted+acetylcholine+receptor+and+sodium+channels.">Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels.</a> Pfl&#252;gers Arch. 407, 577-588 (1986).</li><li>Toro, L., Wallner, M., Meera, P., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maxi-K+(Ca),+Unique+Member+of+the+Voltage-Gated+K+Channel+Superfamily.">Maxi-K (Ca), Unique Member of the Voltage-Gated K Channel Superfamily.</a> News Physiol. Sci. 13, 112-117 (1998).</li><li>Fettiplace, R., Fuchs, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+hair+cell+tuning.">Mechanisms of hair cell tuning.</a> Annu. Rev. Physiol. 61, 809-834 (1999).</li><li>Du, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-sensitive+potassium+channelopathy+in+human+epilepsy+and+paroxysmal+movement+disorder.">Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder.</a> Nat. Genet. 37, 733-738 (2005).</li><li>Ledoux, J., Werner, M. E., Brayden, J. E., Nelson, M. T., T, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-activated+potassium+channels+and+the+regulation+of+vascular+tone.">Calcium-activated potassium channels and the regulation of vascular tone.</a> Physiology (Bethesda). 21, 69-78 (2006).</li><li>Salkoff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-conductance+potassium+channels+of+the+SLO+family.">High-conductance potassium channels of the SLO family.</a> Nat. Rev. Neurosci. 7, 921-931 (2006).</li><li>Shi, J., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+Mg2++enhances+the+function+of+BK-type+Ca2+-activated+K++channels.">Intracellular Mg2+ enhances the function of BK-type Ca2+-activated K+ channels.</a> J. Gen. Physiol. 118, 589-606 (2001).</li><li>Magleby, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gating+mechanism+of+BK+(Slo1)+channels:+so+near,+yet+so+far.">Gating mechanism of BK (Slo1) channels: so near, yet so far.</a> J. Gen. Physiol. 121, 81-96 (2003).</li><li>Latorre, R., Brauchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+conductance+Ca2+-activated+K++(BK)+channel:+activation+by+Ca2++and+voltage.">Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage.</a> Biol. Res. 39, 385-401 (2006).</li><li>Cox, D. H., Cui, J., Aldrich, R. W., W, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+gating+of+a+large+conductance+Ca-activated+K++channel.">Allosteric gating of a large conductance Ca-activated K+ channel.</a> J. Gen. Physiol. 110, 257-281 (1997).</li><li>Cui, J., Aldrich, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+linkage+between+voltage+and+Ca2+-dependent+activation+of+BK-type+mslo1.">Allosteric linkage between voltage and Ca2+-dependent activation of BK-type mslo1.</a> K+ channels. Biochemistry. 39, 15612-15619 (2000).</li><li>Yang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Slo1+BK+channels+by+Mg2++coordinated+between+the+voltage+sensor+and+RCK1.">Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1.</a> 15, 1152-1159 (2008).</li><li>Lee, U. S., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term={beta}+subunit-specific+modulations+of+BK+channel+function+by+a+mutation+associated+with+epilepsy+and+dyskinesia.">{beta} subunit-specific modulations of BK channel function by a mutation associated with epilepsy and dyskinesia.</a> J. Physiol. , 587-1481 (2009).</li></ol>

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. Furth...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Nanoject II Auto-Nanoliter Injector</td>
<td>&nbsp;</td>
<td>Drummond Scientific Company</td>
<td>3-000-204</td>
<td>&nbsp;</td>
<tr>
<td>P-97 Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Perfusion System and Electronic Controller</td>
<td>&nbsp;</td>
<td>AutoMate Scientific, Inc.</td>
<td>ValveLink 16</td>
<td>Inner diameter of perfusion tip: 100 microns</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>CKX31</td>
<td>&nbsp;</td>
<tr>
<td>Glass Pipettes</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>53432-921</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument Co.</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Amplifier</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>AXOPATCH 200B</td>
<td>&nbsp;</td>
<tr>
<td>Computer Interface</td>
<td>&nbsp;</td>
<td>INSTRUTECH Corporation</td>
<td>ITC-18</td>
<td>&nbsp;</td>
<tr>
<td>Headstage</td>
<td>&nbsp;</td>
<td>Axon Instruments</td>
<td>CV 203BU</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

patch clamp and perfusion techniques for studying ion channels expressed in xenopus oocytes

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&deg;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.

Watch this Scientific Journal Video about Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes at JoVE.com

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

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.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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