This work was supported by F31NS086765 (KAC), F31NS076235 (MAP), and R01 NS052669 (GKP) and EIA9100012.&#160; The authors thank Eileen Kasperek for expertise and assistance with molecular biology and tissue culture; and Jason Myers for sharing data obtained from early prefrontal cortical neurons. &#160;

1. Cell Culture and Protein Expression
<ol>
	<li>Maintain HEK293 cells (ATCC number CRL-1573) between passages 22 and 40, which includes passages performed by ATCC, in monolayer culture in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin mixture at 5% CO2 and 37&#160;&#176;C. Between experiments, passage cells into T25 flasks at 5-20 fold dilutions in a final volume of 10 ml. Note: Using cells during these passages guarantees favorable cell health which will allow for optima...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391, 85-100 (1981).</li><li>Piccolino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+electricity+and+the+birth+of+electrophysiology:+the+legacy+of+Luigi+Galvani.">Animal electricity and the birth of electrophysiology: the legacy of Luigi Galvani.</a> Brain Research Bulletin. 46, 381-407 (1998).</li><li>Albright, T. D., Jessell, T. M., Kandel, E. R., Posner, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+Science:+A+Century+of+Progress+and+the+Mysteries+that+Remain.">Neural Science: A Century of Progress and the Mysteries that Remain.</a> Neuron. 25, (2000).</li><li>Popescu, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+glutamate+receptor+gating.">Modes of glutamate receptor gating.</a> The Journal of Physiology. 590, 73-91 (2012).</li><li>Morimoto-Tomita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoinactivation+of+Neuronal+AMPA+Receptors+via+Glutamate-Regulated+TARP+Interaction.">Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction.</a> Neuron. 61, 101-112 (2009).</li><li>Thomas, P., Smart, T. 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In the ion channel field, an important area of research is dedicated to understanding the sequence of events that leads to channel opening or the channel&#8217;s gating mechanism. For most channels, this process is complex and involves several kinetic steps that cannot be deduced from a macroscopic multi-channel signal. In contrast, experiments can be designed where observing the sequence of open/closed events in single channel record can produce more detailed information about gating mechanisms. In the methods described...

Fast communication across biological membranes relies almost exclusively on oligomeric pore forming membrane proteins, commonly referred to as channels. These proteins differ widely in activation signals, gating mechanisms, and conductance properties. Channel proteins whose pores are selective to ions are classified as ion channels; their activation produces ionic currents across the membrane, and their responses can be recorded with high resolution in real time using electrophysiologic techniques. The activation signals span a broad array of chemical and physical inputs including concentration gradients, mechanical and electrical forces, and temperature; thus, further classifying ion channels into ligand gated, mechanosensitive, voltage gated, or heat sensitive types. In this article, protocols are described to record one-channel activity from a ligand gated channel, the NMDA receptor, and a mechanosensitive channel, PIEZO1, using the patch-clamp technique.&#160;
Patch-clamp electrophysiology is the first and most widely used experimental method sufficiently sensitive to permit the observation of single molecules1,2. In addition to this exquisite sensitivity, it has vastly expanded the biological preparations amenable to electrophysiologic recording and also has allowed the observation of ion channels in intact membranes. First, because both voltage clamping and current recording are accomplished with the same electrode, it can be used to record signals across small cells or membranes patches. The technique revealed that ion channels are not restricted to excitable membranes of frog muscles, eel electroplaques, or squid giant axons3,4, but rather that they represent ubiquitous fixtures of transmembrane signaling mechanisms and are intrinsic to all cellular membrane types of uni- or multicellular organisms, and also to intracellular membranes. Importantly, the capability to record transmembrane currents by simply attaching a glass pipette to an intact cell provided the unprecedented opportunity to record activity from ion channels in their native undisrupted membranes. Thus, the cell attached patch-clamp technique, which is described in this protocol, permits monitoring the activity of ion channels continuously for tens of min or longer in their native environment.
Under normal thermal fluctuations, all proteins, including ion channel proteins, undergo structural changes over a broad time scale, with the fastest and most frequent rearrangements represented most likely by side-chain movements and much slower, less frequent changes represented by the repositioning of entire domains or subunits, or in some cases by post translational modifications or protein-protein interactions5,6. Observing long periods of activity generated by one molecule can help to understand the functional dynamics of ion channels in intact physiological membranes and provides valuable information about the operational mechanism of the molecule observed.&#160;
In contrast to the growing understanding of the diversity of ion channels across cell types and developmental stages, knowledge about the molecular composition of ion channels in native membranes is still limited. All ion channels are multimeric proteins and the majority of native ion channels assemble from several types of subunits producing proteins of wide molecular diversity, which is often accompanied with diverse conductance and gating properties. For this reason, ion channels of defined molecular composition are studied upon expression in heterologous systems. In particular, HEK293 cells, which are a clonal line of immortalized human embryonic kidney cells7, gained widespread acceptance as the preferred system for heterologous expression of recombinant ion channels. Among the many advantages that elevated&#160; HEK293 cells as the choice system for ion channel electrophysiology are the ease and affordability of culturing and maintaining long-lived stable cultures, their ability to carry out post-translational folding, processing and trafficking of mammalian proteins, and in many cases, their low level or even absence of endogenous expression for the channel of interest7,8. Expressing recombinant ion channels and studying their functional properties in HEK293 cells continues to be a valuable approach to obtain information about structure-function properties of ion channels as well as the specific properties of ion channel isoforms and their roles in native tissue. The protocols described in this article can be applied equally well to recombinant ion channels expressed in HEK293 cells and to native ion channels.
In summary, the patch-clamp technique, through its unprecedented capacity to resolve signals from one molecule remains, to date, the most direct method for observing the behavior of single molecules. In its cell-attached mode, patch-clamp recording allows long observation periods which, when done for one molecule, can provide exceptional insight into the operation of ion channels. Below is presented a protocol for obtaining high resolution current recordings from cell attached patches containing one ion channel protein.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:189px;">Chemicals</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:128px;">Various</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate Glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bright field inverted microscope</td>
			<td>Olympus</td>
			<td>1x51</td>
			<td>Nikon also has similar microscopes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluroescent box</td>
			<td>X-cite</td>
			<td>Series 120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Light Guide</td>
			<td>X-cite</td>
			<td>OEX-LG15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td colspan="2">Axon Axopatch 200B&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table</td>
			<td>TMC</td>
			<td align="right">63561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIDAQ card</td>
			<td>National Instruments</td>
			<td>776844-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polisher</td>
			<td>Narishige</td>
			<td>Microforge MF-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday Cage</td>
			<td>TMC</td>
			<td align="right">8133306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Pressure Clamp</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA HSPC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressue/Vaccuum Pump</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA PV-PUMP</td>
			<td>For HSPC-1</td>
		</tr>
	</tbody>
</table>

one-channel cell-attached patch-clamp recording

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel&#8217;s conductance properties and the temporal sequence of open-closed conformations that make up the channel&#8217;s activation mechanism, thus helping to understand their functions in health and disease.

Recombinant NMDA Receptors
NMDA receptors bind and respond to the concomitant action of two co-agonists: glutamate and glycine. They assemble as heterotetramers of two glycine binding GluN1 subunits and two glutamate binding GluN2 subunits. GluN2 subunits are encoded by four genes (A-D) and of these the most widely transcribed forms in brain are GluN2A in adult and GluN2B in juvenile animals. Because of the diversity of NMDA receptor subtypes in native preparations, expressing...

Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

One-channel Cell-attached Patch-clamp Recording

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

one-channel-cell-attached-patch-clamp-recording

Research

JoVE Journal

Biology

24.0K visualizações.  University at Buffalo, SUNY.  Descrito aqui é um procedimento para a obtenção de longos trechos de gravação atual de um canal iônico com a técnica de patch-clamp anexado à célula. Este método permite observar, em tempo real, o padrão de open-close conformações dos canais que estão na base do sinal biológico. Estes dados informar sobre as propriedades dos canais nas membranas biológicas não perturbadas. 