이 작품은 KLB와 미국 심장 협회 (09PRE2260209)와 BKM에 아서 J. 슈미트 재단의 사전 박사 장학금으로 국립 심장, 폐, 및 혈액 연구소 (NIH R01-HL089564)의 교부금으로 운용되었다.

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관심 없음 충돌이 선언 없습니다.

여기에서 설명한 방법과 실험 방법은 아주 강력하고 세부 사항에 세심한주의를 적용 할 경우 명확하고 재현 결과를 생성 할 수 있습니다. 좋은 electrophysiological 레코딩 및 수축 / 동맥 세그먼트의 팽창은 각각 셀 및 동맥 세그먼트의 건강에 따라 달라집니다. 셀 준비는 동일한 프로토콜을 사용하여, 날마다 다를 수 있습니다. 절연 솔루션은 최대 2 주까지 사용할 수 있지만 셀 준비의 품질은 두 결과의 일에 낮은 경우 새로운 절연 솔루션이 준비되어야한다. 우리는 효소 활동이 효소의 각각의 새 배치와 최적화를 필요로 일괄 배치되므로 절연 조건 (부화의 시간과 효소의 농도)에 따라 다를 것으로 나타났습니다. 우리는 또한 쥐 장간막 동맥은 여기에 설명 된 세포 격리 프로토콜이 다른 혼합이나 동굴이있을 수 있습니다 다른 혈관 침대 혹은 다른 종에 대한 최적의 있지 않은 것으로 나타났습니다세포 외 기질의 구성 요소 ity. 각 혈관 준비 가장 건강 세포를 생산 조건을 식별 할 수있는 자신의 최적화가 필요합니다. 우리가 건강한 세포를 식별하는 가장 도움이되는 기준은 다음과 같습니다 1) 세포 형태 : 2로 이전 할 때 +, 같은 모양을 유지 50 μM 칼슘이 포함 된 절연 솔루션에 MA 세그먼트를 triturating 후, 부드러운 가늘고 긴하지만, 약간 죄 모습 MM 칼슘 2 + 함유 외부 솔루션입니다. 모든 세포는 거의 완벽하게 편안한 가늘고 긴 모양을 유지됩니다 만 거의 완전히 완화 근세포는 electrophysiological 레코딩에 사용되어야한다, 2) 세포가 손상 손상 멤브레인을 나타내는, 광학 조밀해야, 3) 건강 근세포의 휴식 막 전압에 사용 기록 칼륨 전류 또는 막 전압의 전류 클램프 녹음 용 (보통 이온 조건 데에 따라 -56 뮤직 비디오에 -45 mV의 범위에서 -40 뮤직 비디오보다 더 부정적인해야) 여기 프로토콜에 cribed. 성공 electrophysiological 녹음, 목욕 솔루션과 피펫 솔루션 (구성에 대한 표 1 참조) 실험 당일 준비해야합니다. 보장하기 위해 그것은 (298-299 mOsM) 동일이 될 수있는 내부 및 욕조 솔루션의 osmolality를 조정하는 데있어 매우 중요합니다. 더 자세히 대략 isometric 힘을 측정보다 생리 조건을 압력 myography를 사용하여 작은 동맥 (&lt;500 μm)에서 만든 Isobaric 동맥 기능 측정 와이어 myograph를 사용했다. 압력 myograph의 선박은 일반적으로보다 밀접하게 생체 5, 6으로 측정 민감한를 건수 혈관 수축 제라 agonists 2-4, 더 민감합니다. agonists의 낮은 농도를 증가 감도는 AVP의 생리 학적 관련 picomolar 농도에 의해 유도 신호 전달 메커니즘의 식별을 사용하고 있습니다7, 8. 압력 myography은 혈관의 구조를 보존하고 내피의 무결성을 유지합니다. 약물의 내피로 인한 효과는 온전하고 내피 - denuded 선박 9 병렬 측정을 실시하여 평활근로 인한 효과를 구별 할 수 있습니다. 내피의 의도적 방해가 물리적으로 내피을 (루멘을 통해 앞뒤로 인간의 머리카락을 전달하여 예) 손상 또는 동맥의 내강을 통해 공기를 관류하여 수행 할 수 있습니다. 내피 기능 (하지만, 혈관하지 평활근 기능)의 손실이 작용제 9 미리 죄 선박에서 아세틸 콜린에 대한 할인 내피로 인한 vasodilator 응답을 측정하여 확인해야합니다. 패치 클램프 기술과 동맥 수축 / 창 자간 막 arteri의 팽창을 사용하여 창 자간 막 동맥 근세포의 이온 전류 / 막 전압의 병렬 측정압력 myography를 사용하여 에스는 생리적 신호 전달 폭포 모두와 치료 대리인의 행동에서 특정 이온 채널의 역할 해설을 활성화 할 수 있습니다. 이온 채널 또는 특정 신호 전달 중간체의 특정 클래스를 타겟으로 치료 세포 수준에서 myocyte 함수의 규제 및 수축 / 그대로 동​​맥의 팽창에 이러한 채널의 역할에 대한 기계론의 정보를 제공하기 위해 additively 또는 순서에 따라 적용 할 수 있습니다. 동맥 직경에 해당하는 효과를 이온 전류의 특정 유형을 강화하거나 억제 트리트먼트 수렴 결과는 자체적으로 두 가지 방법을 사용하여 얻을 수보다 더 신뢰할 수있는 해석을 제공합니다. 이상적으로, 연구에 따라 에이전트의 농도 - 의존성은 두 시스템에서 결정해야합니다. 생리학 또는 치료 관련성을 평가하기 위해 테스트 농도는 생리적 환경 O로 측정 농도에 관한해야R은 임상 치료와 함께 달성했습니다. 실험 방법 이러한 유형의 예는 이전 출판물 8-10에서 찾을 수 있습니다.

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> 나트륨 염화물 </td><td> 시그마 </td><td> S5886 </td><td> 해부 솔루션 : 145 전기 생리학에 대한 목욕 솔루션 * : 140 전기 생리학에 대한 내부 솔루션 : 10 근세포에 대한 절연 솔루션 * : 140 압력 myography을위한 목욕 솔루션 : 145 압력 myography에 대한 루멘 솔루션 : 145 </td></tr><tr><td> 염화칼륨 </td><td> 시그마 </td><td> P5405 </td><td> 해부 솔루션 : 4.7 전기 생리학에 대한 목욕 솔루션 * : 5.36 전기 생리학에 대한 내부 솔루션 : 135 근세포에 대한 절연 솔루션 * : 5.36 압력 myography을위한 목욕 솔루션 : 4.7 압력 myography에 대한 루멘 솔루션 : 4.7 </td></tr><tr><td> 칼륨 EGTA </td><td> 시그마 </td><td> E4378 </td><td> 전기 생리학에 대한 내부 솔루션 : 0.05 </td></tr><tr><td> HEPES </td><td> 시그마 </td><td> H9136 </td><td> 전기 생리학에 대한 목욕 솔루션 * : 10 전기 생리학에 대한 내부 솔루션 : 10 근세포에 대한 절연 솔루션 * : 10 </td></tr><tr><td> 나트륨 수소 인산염 </td><td> 시그마 </td><td> S5136 </td><td> 근세포에 대한 절연 솔루션 * : 0.34 </td></tr><tr><td> 칼륨 수소 인산 </td><td> 시그마 </td><td> P5655 </td><td> 근세포에 대한 절연 솔루션 * : 0.44 </td></tr><tr><td> 염화 마그네슘 </td><td> 시그마 </td><td> M2393 </td><td> 전기 생리학에 대한 목욕 솔루션 * : 1.2 전기 생리학에 대한 내부 솔루션 : 1 근세포에 대한 절연 솔루션 * : 1.2 </td></tr><tr><td> 칼슘 염화물 </td><td> 시그마 </td><tD&gt; C7902 </td><td> 전기 생리학에 대한 목욕 솔루션 * : 2 근세포에 대한 절연 솔루션 * : 0.05 </td></tr><tr><td> 인산 나트륨 </td><td> 피셔 과학 </td><td> BP331-1 </td><td> 해부 솔루션 : 1.2 압력 myography을위한 목욕 솔루션 : 1.2 압력 myography에 대한 루멘 솔루션 : 1.2 </td></tr><tr><td> 황산 마그네슘 </td><td> 시그마 </td><td> M2643 </td><td> 해부 솔루션 : 1.17 압력 myography을위한 목욕 솔루션 : 1.17 압력 myography에 대한 루멘 솔루션 : 1.17 </td></tr><tr><td> 걸레 </td><td> 피셔 과학 </td><td> BP308 </td><td> 해부 솔루션 : 3 압력 myography을위한 목욕 솔루션 : 3 압력 myography에 대한 루멘 솔루션 : 3 </td></tr><tr><td> Pyruvic 산 </td><td> 시그마 </td><td> P4562 </td><td> 해부 솔루션 : 2 에 대한 목욕 솔루션압력 myography : 2 압력 myography에 대한 루멘 솔루션 : 2 </td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) 이수화 </td><td> 연구 유기물 </td><td> 9572E </td><td> 해부 솔루션 : 0.02 압력 myography을위한 목욕 솔루션 : 0.02 압력 myography에 대한 루멘 솔루션 : 0.02 </td></tr><tr><td> D-포도당 </td><td> 시그마 </td><td> G7021 </td><td> 해부 솔루션 : 5 전기 생리학에 대한 목욕 솔루션 * : 10 전기 생리학에 대한 내부 솔루션 : 20 근세포에 대한 절연 솔루션 * : 10 압력 myography을위한 목욕 솔루션 : 5 압력 myography에 대한 루멘 솔루션 : 5 </td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A3912 </td><td> 해부 솔루션 : 1 % 압력 myography에 대한 루멘 솔루션 : 1 % </td></tr><tr><td> 피에이치 </td><td></td><td></td><td> Solutio을 해부N : 7.4 전기 생리학에 대한 목욕 솔루션 * : 7.3 전기 생리학에 대한 내부 솔루션 : 7.2 근세포에 대한 절연 솔루션 * : 7.2 압력 myography을위한 목욕 솔루션 : 7.4 압력 myography에 대한 루멘 솔루션 : 7.4 </td></tr><tr><td> Osmolarity </td><td></td><td></td><td> 해부 솔루션 : 300 전기 생리학에 대한 목욕 솔루션 * : 298 전기 생리학에 대한 내부 솔루션 : 298 근세포에 대한 절연 솔루션 * : 298 압력 myography을위한 목욕 솔루션 : 300 압력 myography에 대한 루멘 솔루션 : 300 </td></tr><tr><td></td><td></td><td></td><td> * 11 </td></tr><tr><td></td><td></td><td></td><td> 표 1. 실험에 사용되는 솔루션의 구성 요소. </td></tr></tbody></table>

exploring arterial smooth muscle kv7 potassium channel function using patch clamp electrophysiology and pressure myography

저항 동맥의 벽 내의 평활근 세포의 수축이나 이완이 동맥 직경을 결정함으로써 혈관을 혈액의 흐름을 제어하고 조직 혈압에 기여한다. 수축 과정은 주로 이온 수송 및 채널의 다양한 의해 제어 차례에 cytosolic 칼슘 농도 ([칼슘 2 +] cyt)에 의해 조절된다. 이온 채널은 혈관 수축 또는 vasodilation에 영향을 미치도록 vasoactive 호르몬에 의해 활성화 신호 전달 경로의 일반적인 중간체 있습니다. 그리고 이온 채널은 종종 두 의도적으로 치료 대리인 또는 실수 (원치 않는 심장 혈관 부작용을 유발하는 등) (예 : 칼슘 채널 차단제가 vasodilation과 낮은 혈압을 유도하기 위해 사용)에 의해 타겟팅 할 수 있습니다. Kv7 (KCNQ) 전압 활성화 칼륨 채널은 최근에 중요한 생리 및 치료 targ으로 연루되어있다부드러운 근육 수축의 조절을위한 ETS. 생리적 신호 전달 모두와 치료 대리인의 행동에 Kv7 채널의 특정 역할을 명료하게하다하기 위해, 우리는뿐만 아니라 그대로 동​​맥의 맥락에서의 공헌을 평가로서 활동이 세포 수준에서 조절이 얼마나 공부를해야합니다. 쥐 창 자간 막 동맥 유용한 모델 시스템을 제공합니다. 동맥은 쉽게 해부하는 결합 조직의 청소 및 패치 클램프 전기 생리학에 대한 격리 된 동맥 근세포를 준비하는 데 사용하거나, cannulated 상대적으로 생리적 조건 하에서 혈관 수축 제라 / vasodilator 응답 측정을 위해 가압 될 수 있습니다. 여기 측정의 두 가지 유형에 사용되는 방법을 설명하고 실험 디자인 혈관 톤의 규정에 이러한 이온 채널의 역할을 명확하게 이해를 제공하기 위해 통합 될 수있는 방법의 예를 제공합니다.

Watch this Scientific Journal Video about Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography at JoVE.com

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

압축시키는 것 / 확장시키는 반응 (압력 myography를 사용하여)의 측정 병렬 절연 동맥 근세포 (패치가 el​​ectrophysiological 기술 클램프 사용)에서 Kv7 (KCNQ) 칼륨 채널 활동의 측정은 혈관 평활근의 생리학에 Kv7 채널의 역할에 대한 중요한 정보를 공개 할 수 있습니다 약리학.

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 ...

exploring-arterial-smooth-muscle-kv7-potassium-channel-function-using

Nanyang Technological University

Research

JoVE Journal

Biology

15.1K 조회수.  Loyola University Chicago. 압축시키는 것 / 확장시키는 반응 (압력 myography를 사용하여)의 측정 병렬 절연 동맥 근세포 (패치가 el​​ectrophysiological 기술 클램프 사용)에서 Kv7 (KCNQ) 칼륨 채널 활동의 측정은 혈관 평활근의 생리학에 Kv7 채널의 역할에 대한 중요한 정보를 공개 할 수 있습니다 약리학.

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

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

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

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 ...

Focal Ca2+ Transient Detection in Smooth Muscle

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ from internal stores, and allows multiple cells to be monitored simultaneously to assess, for example, coupling in syncytial tissue. Subcellular Ca2+ transients are common in smooth muscle, yet are difficult to measure accurately because of the problems caused by their stochastic occurrence, over an often wide field of view, in an organ that it prone to contract. To overcome this problem, we've developed a series of imaging protocols and analysis routines to acquire and then analyse, in an automated fashion, the frequency, location and amplitude of such events. While this approach may be applied in other contexts, our own work involves the detection of local purinergic Ca2+ transients for locating transmitter release with submicron resolution. 
ATP is released as a cotransmitter from autonomic nerves, where it binds to P2X1 receptors on the smooth muscle of the detrusor and vas deferens. Ca2+ enters the smooth muscle, resulting in purinergic neuroeffector Ca2+ transients (NCTs). The focal Ca2+ transients allow the optical monitoring of neurotransmitter release in a manner that has many advantages over electrophysiology. Apart from the greatly improved spatial resolution, optical recording has the additional advantage of allowing the recording of transmitter release from many distinguishable sites simultaneously. Furthermore, the optical plane of focus is easier to maintain or correct during long recording series than is the repositioning of an intracellular sharp microelectrode. 
In summary, a method for imaging of Ca2+ fluorescence is outlined which details the preparation of tissue, and the acquisition and analysis of data. We outline the use of several scripts for the analysis of such Ca2+ transients.

Focal Ca2+ Transient Detection in Smooth Muscle

Details methods for high-resolution Ca2+ imaging of smooth muscle within isolated organs, including: preparation of the tissue, image acquisition and data analysis.

초점 칼슘 2 +</sup평활근에서> 과도 감지

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

인간의 탯줄 아티 평활근 세포 절연 (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

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 ...

Assessing Murine Resistance Artery Function Using Pressure Myography

Pressure myograph systems are exquisitely useful in the functional assessment of small arteries, pressurized to a suitable transmural pressure. The near physiological condition achieved in pressure myography permits in-depth characterization of intrinsic responses to pharmacological and physiological stimuli, which can be extrapolated to the in vivo behavior of the vascular bed. Pressure myograph has several advantages over conventional wire myographs. For example, smaller resistance vessels can be studied at tightly controlled and physiologically relevant intraluminal pressures. Here, we study the ability of 3rd order mesenteric arteries (3-4 mm long), preconstricted with phenylephrine, to vaso-relax in response to acetylcholine. Mesenteric arteries are mounted on two cannulas connected to a pressurized and sealed system that is maintained at constant pressure of 60 mmHg. The lumen and outer diameter of the vessel are continuously recorded using a video camera, allowing real time quantification of the vasoconstriction and vasorelaxation in response to phenylephrine and acetylcholine, respectively.
	To demonstrate the applicability of pressure myography to study the etiology of cardiovascular disease, we assessed endothelium-dependent vascular function in a murine model of systemic hypertension. Mice deficient in the &alpha;1 subunit of soluble guanylate cyclase (sGC&alpha;1-/-) are hypertensive when on a 129S6 (S6) background (sGC&alpha;1-/-S6) but not when on a C57BL/6 (B6) background (sGC&alpha;1-/-B6). Using pressure myography, we demonstrate that sGC&alpha;1-deficiency results in impaired endothelium-dependent vasorelaxation. The vascular dysfunction is more pronounced in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice, likely contributing to the higher blood pressure in sGC&alpha;1-/-S6 than in sGC&alpha;1-/-B6 mice. 
	Pressure myography is a relatively simple, but sensitive and mechanistically useful technique that can be used to assess the effect of various stimuli on vascular contraction and relaxation, thereby augmenting our insight into the mechanisms underlying cardiovascular disease.

In pressure myography, an intact small segment of a vessel is mounted onto two small cannulas and pressurized to a suitable luminal pressure. Here, we describe the method to measure vasorelaxation response of the mouse 3rd order mesenteric arteries in c57 and sGC&alpha;1-/- mice using pressure myography.

압력 Myography를 사용 쥐과 저항 동맥의 기능을 평가

Pressure myograph systems are  exquisitely useful in the functional assessment of small arteries, pressurized  to a suitable transmural pressure. The  ...

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 ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

약리학 연구에 고립 된 조직 목욕-애플리케이션에서 부드러운 근육 기능의 측정

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.