압력 연마 기법은 SR의 Lockery와 공동으로 개발되었습니다.

풀링 pipettes 풀러와 같은 필라멘트를 사용하면 셔터는 P - 97 / 브라운 Microelectrode의 풀러 플라밍 약 10-20 pipettes 세트를 가져옵니다. <ol><li> 당신의 유리를 선택합니다. 우리는 borosilicate을 (BF150 - 86 - 10, OD = 1.5 mm, ID = 0.86 mm)을 사용합니다. 그것이 깨끗하고 먼지가 무료로 남아 있도록 신중하게 유리를 저장합니다. </li><li> 당기는 프로그램을 디자인합니다. 기본 목표는 응용 프로그램에 따라 달라집니다 약 원하는 크기를 여는 팁입니다. 서터의 P - 97 피펫 요리책 2 훌륭한 참조입니다. </li><li> 개방 직경과 매끄러운를 결정하는 현미경 아래 pipettes을 관찰. 거친 고르지 또는 불규칙 조언을​​ 폐기하십시오. 표준 패치 클램프 녹음, 전반 2-4 μm의하는 팁 오프닝은 최고입니다. 무딘 팁은 선호하는 것과 동일한 오프닝 직경에 대한 저항을 낮은 발생합니다. 모양과 크기는 아래 연마하는 동안 수정할 수 있습니다. </li></ol> 압력 연마 pipettes <ol><li> 깨끗한 압축 공기 라인에 피펫 홀더를 연결합니다. 레귤레이터를 사용하여 ~ 40 PSI로 압축 공기 라인 압력을 설정합니다. 피펫 홀더 압력 및 해제할 수 수있는 4 방향 제어 밸브에 압축 공기 라인을 연결합니다. Tygon의 ⅛의 길이 ® 튜브와 제어 밸브에 피펫 홀더를 연결합니다. </li><li> 고압 피펫 홀더, 피트 페달, 낮은 높은 배율, 오랜 실무 거리 (이상, 10X와 100x) 공기 목표 장착된 현미경에 의해 통제 연마 코일과 연마 장비 (microforge)를 사용하십시오. 이것은 프로토콜 동안 피펫 오픈을 관찰 수 있도록 중요합니다. </li><li> 옵션 : 코트 용량을 감소하고 (이 기술에 대해 &quot;풀링 pipettes&quot;참조) 잡음 특성을 개선하기 위해 소수성 절연체로 피펫. </li><li> 압력 밸브가 OFF 위치에 있는지 확인합니다. 피펫 홀더에 피펫을 놓고 손가락으로 꽉 맞는를 조입니다. 안전주의 : 피펫이 피펫 홀더에 단단히 개최하는 경우, 공기 압력은 피펫이 피펫 홀더에서 배출되는 원인이됩니다. </li><li> microforge의 피펫과 홀더를 놓으십시오. 현미경 단계에 위치 피펫 홀더 너무 팁 및 연마 코일 모두 microforge 부족 (10X) 전원에서 볼 수있다. ~ 75 미크론 필라멘트의 끝부분을 가져와. 가열하면 필라멘트가 확장됩니다 유의하십시오. </li><li> 높은 전력 (100x) 목표로 전환합니다. 피펫 팁 및 연마 코일 모두 초점과 전망 분야에서해야합니다. 그렇지 않다면, 연마 코일의 높이를 조정합니다. (피펫의 위치를​​ 조정할 수 없습니다.) 피펫 팁 및 연마 코일 볼 분야의 반대편에 가까운해야합니다. 그것은 가열로 확장하여 피펫 뛰어들고 있기 때문에 너무 피펫 팁 가까운 코일을 위치하지 마십시오. </li><li> 피펫으로 압축 공기를 직접하기 위해 밸브를 돌려과 팁 뒤에 테이퍼가 바깥쪽을 풍선 시작 때까지 피펫 팁을 열. </li><li> 끝 너무 광범위하게 풍선 전에 가열을 중지합니다. 실험은 원하는 형태에 해당하는 거리, 열, 시간을 결정하기 위해 필요합니다. </li><li> 옵션 : 압력의 부재에있는 팁을 연마하여 팁 오프닝 직경을 절감할 수 있습니다. 단계 7과 8은 조정 피펫 팁의 형상과 크기 모두를 반복하실 수 있습니다. </li><li> 압력을 끄고 안전한 방향으로 피펫 팁을 지적하면서, 피펫을 제거하고 그것 분진으로부터 보호됩니다 깨끗한 저장 상자에 넣습니다. 안전주의 : 공기 압력이 해제되지 않은 경우, 피펫가 제거하는 동안 피펫 홀더에서 꺼낼 수 있습니다. </li></ol> microforge에 대한 참고 사항 우리는 재고품의 상용 부품의 조립 microforge을 사용합니다. (당신은 또한 몇몇 기업, 예를 들어, 에이러 과학, GlassWorx에서 압력 연마 키트를 구입할 수 있습니다.) 상관없이 microforge의 그것은 주로 직경 단지 몇 μm의입니다 팁 오프닝을 볼 승산이 충분 한가하는 광학을 가지고하는 것이 필수적입니다. 우리는이 목적을 위해 긴 작동 거리 100x 렌즈를 사용합니다. 또한, 코트 위해 높은 용해 온도 유리 연마 코일은 pipettes에서 증발 금속을 입금 최소화하기 위해. 파트 3 : 예상 결과 보통 압력 연마 직경을 열에 감지할 수있을 정도의 변화없이 피펫의 저항을 줄일 수 있습니다. 원하는 경우,이 프로토콜은 5 10MΩ 사이의 서브 마이크론 채용 및 resistances와 pipettes를 만들기 위해 사용할 수 있습니다.

<ol>
	<li>Goodman, M. B., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+polishing:+a+method+for+re-shaping+patch+pipettes+during+fire+polishing. .">Pressure polishing: a method for re-shaping patch pipettes during fire polishing. .</a> J Neurosci Methods. 100, 13-15 (2000).</li></ol>

우리는 원래 C로 2-3 μm의 뉴런에서 패치 - 클램프 녹음 pipettes 유용하게이 방법을 개발 엘레간스 1. 그것은 굿맨 실험실에서 일상 사용할 수 있습니다. 작은 C.에서뿐만 아니라, 레코딩을위한 우리 압력 폴란드어 pipettes 엘레간스의 뉴런뿐만 아니라 Xenopus oocytes의 표현 이온 채널의 macropatch 및 단일 채널 녹음을 얻는. 주어진 팁 - 오프닝 지름은 뭉툭한 형태는 낮은 저항 피펫을 생?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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>Glass Capillaries</td>
<td>Consumable</td>
<td>Sutter Instruments</td>
<td>BF150-85-10 (or similar)</td>
<td>The method works with any type of capillary except those made from quartz. However, we recommend using capillaries with a filament.</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>Microscope</td>
<td>Leica</td>
<td>DM IL (or similar)</td>
<td>Any simple inverted microscope will do. However, it is essential to have sufficient magnification to see pipettes during polishing. We use a 100x metallurgical lens with a long working distance for this purpose.</td>
</tr>
<tr>
<td>Microforge Kit</td>
<td>Tool</td>
<td>ALA Science</td>
<td>CPM-2</td>
<td>Supplies polishing coil, controller and foot pedal.</td>
</tr>
<tr>
<td>Air pressure source</td>
<td>Tool</td>
<td>ALA Science</td>
<td>PR-60</td>
<td>Accessory for providing high pressure, includes a suitable valve. Alternatively, compressed air from the building can be used together with a filter/regulator (set to 40 PSI) and a 4-way valve (see below)</td>
</tr>
<tr>
<td>4-way valve</td>
<td>Tool</td>
<td>Cole-Parmer</td>
<td>A-98150-01</td>
<td>Fittings will be needed to couple valve to tubing</td>
</tr>
<tr>
<td>Pipette Holder with Luer Connector</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH-6S20</td>
<td>Use a male luer-to-barb fitting to connect pipette holder to high-pressure tubing</td>
</tr>
<tr>
<td>Tygon&#174; tubing</td>
<td>Tool</td>
<td>Cole-Parmer</td>
<td>06408-62</td>
<td>Use this tubing to connect pipette holder to 4-way valve
</td>
</tr>		</tbody>
		</table>
		</div>

pressure-polishing pipettes for improved patch-clamp recording

압력 연마는 패치 - 클램프 녹음 유리 pipettes을 형성하는 방법이다. 우리가 먼저 작은 (&lt;3m)의 연결을 세포 기관에서 녹화에 적합한 pipettes를 fabricating이 방법을 개발했습니다. 기본 교장 유리 불어와 유사하며 일반 micropipette의 풀러 준비한 패치 pipettes의 모양을 수정하는 공기 압력과 열을 결합되어 있습니다. 이것은 소위 소프트 (소다 석회)와 하드 (borosilicate) 안경에 적용할 수 있습니다. 일반적으로 압력 연마는 팁 오프닝 (굿맨과 Lockery, 2000)의 직경을 감소하지 않고 25 % 피펫 저항을 줄일 수 있습니다. 이것은 유리의 거의 모든 유형에 적용 고압 밸브와 microforge에 맞는만을 추가 요구하실 수 있습니다. 이 기술은 ultrasmall 세포 (&lt;5m)에서 녹음을 위해 필수적이며, 또한 피펫의 저항을 최소화하여 단일 채널 녹음을 향상시킬 수 있습니다. 무딘 모양이 천공 - 패치 클램프 녹음에도 유용합니다 천공에 사용할 수있는 큰 막 물집이 끝 모양 결과 때문이다.

Watch this Scientific Journal Video about Pressure-polishing Pipettes for Improved Patch-clamp Recording at JoVE.com

Pressure-polishing Pipettes for Improved Patch-clamp Recording

이것은 유리 micropipettes의 모양을 수정하는 가이드입니다. 특히, 열 및 공기 압력을 사용하여 테이퍼가 낮은 피펫 저항을 선도 팁 오프닝을 증가하지 않고 확대됩니다. 이것은 작은 세포의 낮은 소음 녹음을 얻는 것이 중요합니다하지만 많은 응용 프로그램에서 유용합니다.

향상된 패치 - 클램프 녹음 압력 연마 Pipettes

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

pressure-polishing-pipettes-for-improved-patch-clamp-recording

Research

JoVE Journal

Biology

12.9K 조회수.  Stanford University School of Medicine. 이것은 유리 micropipettes의 모양을 수정하는 가이드입니다. 특히, 열 및 공기 압력을 사용하여 테이퍼가 낮은 피펫 저항을 선도 팁 오프닝을 증가하지 않고 확대됩니다. 이것은 작은 세포의 낮은 소음 녹음을 얻는 것이 중요합니다하지만 많은 응용 프로그램에서 유용합니다.

비디오: Pressure-polishing Pipettes for Improved Patch-clamp Recording

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

칼리에서 패치 클램프 커패시턴스 레코딩을위한 방법

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

연구

생물학

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oocytes의 표현 이온 채널 패치 클램프 녹음

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

프로그래밍 풀러와 패치 - pipettes와 샤프 전극을 만들기

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

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.

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.

이온 채널 유학을위한 패치 클램프 및 재관류 기법으로 표현 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 ...

Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ubiquity of microbes creates a copious supply of potential contaminants in a laboratory. To ensure experimental success, the number of contaminants on equipment and work surfaces must be minimized. Common among many experiments in microbiology are techniques involving the measurement and transfer of cultures containing bacterial cells or viral particles. To do so without contacting non-sterile surfaces or contaminating sterile media requires (1) preparing a sterile workspace, (2) precisely setting and accurately reading instruments for aseptic transfer of liquids, and (3) properly manipulating instruments, cultures flasks, bottles and tubes within a sterile field. Learning these procedures calls for training and practice. At first, actions should be slow, deliberate, and controlled with the goal being for aseptic technique to become second nature when working at the bench. Here we present the steps for measuring volumes using serological pipettes and micropipettors within a sterile field created by a Bunsen burner. Volumes range from microliters (&mu;l) to milliliters (ml) depending on the instrument used. Liquids commonly transferred include sterile broth or chemical solutions as well as bacterial cultures and phage stocks. By following these procedures, students should be able to: &#8226;Work within the sterile field created by the Bunsen burner flame. &#8226;Use serological pipettes without compromising instrument sterility.&#8226; Aspirate liquids with serological pipettes, precisely reading calibrated volumes by aligning the meniscus formed by the liquid to the graduation marks on the pipette. &#8226;Keep culture bottles, flasks, tubes and their respective caps sterile during liquid transfers. &#8226;Identify different applications for plastic versus glass serological pipettes. &#8226;State accuracy limitations for micropipettors. &#8226;Precisely and accurately set volumes on micropipettors. &#8226;Know how to properly use the first and second stop on a micropipettor to aspirate and transfer correct volumes.

When working in a laboratory, it is imperative to minimize sources of contamination. Aseptic technique refers to procedures that permit transfer of cultures and reagents while avoiding contact with non-sterile surfaces. Serological pipettes and micropipettors are used to measure precise volumes without compromising sterility of solutions used in experiments.

무균 실험실 기술 : Serological Pipettes 및 Micropipettors로 볼륨 이동

Microorganisms are everywhere - in the air, soil, and human body as well as on inanimate surfaces like laboratory benches and computer keyboards. The ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

패치 클램프 전기 생리학 및 압력 Myography를 사용하여 간선 평활근 Kv7 칼륨 채널 기능을 탐색

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

이미징 및 패치 클램프 전기 생리학에 대한 거대한 리포좀 준비

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

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.

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.

1 채널의 세포 부착 된 패치 클램프 녹음

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

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

횡단 단백질의 재구성, 현미경 및 패치 클램프 연구 거대한 단일 층 소포에 전압 게이트 이온 채널, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

패치 클램프 및 라이브 형광 현미경을 구현 갓 고립 PKD 상피 세포의 기능적 특성을 모니터링하려면

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...