KISEP Otology Korean J Otolaryngol 2002;45:550-6 기니픽내이의 Deiters 세포에서측정된외향정류성칼륨이온전류 김효준 1 안중호 2 김원태 3 임채헌 3 정종우 2 이광선 2 Outward Rectifying Current in Isolated Deiters Cells from Guinea Pig Cochlea Hyo Joon Kim, MD 1, Joong Ho Ahn, MD 2, Won Tae Kim, MS 3, Chae Hun Leem, MD 3, Jong Woo Chung, MD 2 and Kwang-Sun Lee, MD 2 1 Graduate School, University of Ulsan, 2 Department of Otolaryngology and 3 Physiology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea ABSTRACT Background and ObjectivesThe Deiters cell is one of the supporting cells in the organ of Corti and is known to possibly regulate the signal transduction pathway in the organ of Corti. The signal transduction process can be modulated by ATP and acetylcholine, the so-called neurotransmitters, in Deiters cells. Intracellular Ca 2 concentration can be also increased by these neurotransmitters and the control mechanism on the organ of Corti is highly suggested in Deiters cells. Potassium ion K + is known to be important both in hair cells and supporting cells. Through K + channel, the membrane potential may be controlled and the signal transduction pathway can be regulated. Furthermore, the motility of outer hair cell and the signal transduction from the apical stereocilia are considered to be regulated by this channel. The aim of this study is to record the K + current in the isolated Deiters cells from guinea pig cochlea. Materials and MethodsDeiters cells were isolated from the organ of Corti of guinea pig by using collagenase and a pipet. A whole cell patch clamp was performed under the inverted microscope and the current was measured with List-7 amplifier and pclamp 8.0.2 software. ResultsThe resting membrane potential was 15.022.66 mv n6. When the cell membrane was hyperpolarized into 110 mv from the 40 mv holding potential, the peak current was 22739.9 pa n15. After having depolarized to the maximum, 50 mv, the peak current was 7123737 pa, and the reversal potentials of different external K + concentration changed in the K + -dependent manner. About 80 of this current was inhibited by TEA. When K + was substituted by Cs +, the peak current was 1788231 pa at 50 mv step pulse. Activation curve of this outward current showed two different Vh half activation voltage and K slope factor. ConclusionOutward rectifying K + channels exist in Deiters cells and they can be inhibited by TEA and permeable to Cs +. More than two types of K + current can exist and they may play a role in the recovery of membrane potential after depolarization. Korean J Otolaryngol 2002;45:550-6 KEY WORDSPotassium channels Organ of corti Labyrinth supporting cells Patch-Clamp techniques Signal transduction. 550
김효준 외 조절할 수 있는 것으로 추정되고 있다. 즉, 지지세포인 Deiters 세포, Hensen 세포, pillar 세포 등에 신경접합이 존재 3-5) 한다고 보고 되었고, 이들 세포가 신경전달물질인 ATP 6-10) 나 아세틸콜린에 대한 세포내의 반응이 보고 되어 점차 실정이다. 본 연구에서는 기니픽 내이 코티기관에서 분리된 Deiters 세포의 세포막 전류를 측정하여 Deiters 세포에 존재하는 이 온통로에 대해 알아보고자 하였다. 지지세포에 대한 관심이 고조되고 있다. 재료 및 연구방법 이중 코티기관 지지세포 중 비교적 많은 연구가 이루어진 Deiters 세포는 대뇌로부터 원심성 신경섬유 접합이 세포기 저부에 분포하며,7)8) 원심성 신경전달물질로 여겨지는 아세틸 2 콜린과 ATP에 대하여 세포내 Ca Deiters 세포의 분리 농도가 증가되며 이는 백색 기니픽(200~250 g)에 sodium pentobarbital(50 산화질소/환식구아노신 모노포스페이트(cyclic GMP) 경로를 mg/kg)을 복강내 주사하여 마취시킨 후 양측 측두골을 얻 통하여 억제되는 조절기전을 가지고 있다.9) 더욱이 Deiters 었다. Deiters 세포의 분리는 1형 collagenase(sigma, St. 세포에 존재하는 아세틸콜린 수용체는 외유모세포에서 발견 Louis, MO, USA) 1 mg을 정상 Tyrode 용액 1 ml에 녹인 되는 것과 같은 α-9형의 수용체로 알려져 외유모세포에서 후 0.5 mg의 최종농도로 상온에서 20 분간 소화시켜 얻어 와 같은 신호전달기전이 Deiters 세포내에 존재할 가능성을 냈다. 분리된 세포를 50 μl의 정상 Tyrode 용액이 포함된 높여주고 있다.10)11) 특히 외유모세포에 접하여 있고, 세포내 35 mm 배양용기에 넣어 약 20분간 세포가 바닥에 붙도록 2 농도의 증가로 인한 phalangeal process의 움직임이 기다린 후 분리된 세포 중 세포의 다각형 모양이며 긴 pha- 보고 되어 외유모세포에 대한 물리적인 조절기전의 가능성이 langeal process를 가지는 특징적인 모양의 Deiters 세포를 제기되고 있다. 그러므로 Deiters 세포에 의한 외유모세포의 확인한 후 실험하였다(Fig. 1). Ca 조절은 청각신호전달과정에서 중요한 조절기전으로 작용할 것으로 추정할 수 있다. 막전압고정법 이온통로는 세포의 신호전달에 중요한 역할을 하며, 이를 Deiters 세포가 들어있는 현탁액을 실험 용기에 옮겨 도 통한 이온의 흐름에 따라 막전압을 변화시키고 세포내 2차 립현미경(Olympus LH50A, Olympus Optical Ins LTD, 신호전달계를 활성화 시키며 조절한다. 이러한 이온통로는 와 Japan)시야에서 실험하였다. 도립현미경 상부에 위치한 용 우로 들어오는 물리적인 진동에너지를 전기신호로 바꾸어 주 량 0.2 ml 정도의 실험용기에 세포가 함유된 저장용액을 는 역할을 하며, 외유모세포에서는 주파수 특이적인 반응을 떨어뜨린 후 세포들이 실험용기에 부착하도록 10분동안 기 유발하는 역할을 한다. 그러므로 코티기관내의 Deiters 세포 다렸다. 칼슘이온이 함유된 정상 Tyrode 용액을 계속 흘 에 존재하는 이온통로를 밝히고 이의 특성을 이해하는 것은 려 관류시켰으며 관류 속도는 2 ml/min로 하였고 용액 온 이 세포내의 신호전달기전을 이해하게 됨과 동시에 코티기관 도를 37 로 유지하였다. 적당한 세포를 고른 후 400배 시 의 조절작용을 이해하여 청각신호전달과정을 파악하는데 도 야에서 미세조작기에 연결된 전극을 세포에 접근, 접촉시 12) 움을 준다. Nenov등 은 막전압고정법을 이용하여 Deiters 세포에서 K 선택적인 외향 정류성 전류를 기록하였으며, 이 는 Kv1.5 이온통로와 유사한 형이라고 하였고, 전압의존성 Ca2 전류와 비선택성 전류는 기록하지 못하였다. Deiters 세 포도 외유모세포와 마찬가지로 세포의 대부분은 외림프액 내 에 존재하지만 phalangeal process의 일부는 내림프액 내 에 면해있으며, 내림프액의 K 농도는 외림프액과는 정반대 로 약 150 meq/l 정도이다. 그러므로 Deiters 세포내에 K 통로가 존재하여 K 농도경사에 따른 생리학적인 역할을 담 당할 가능성이 매우 높다. 또한 Deiters 세포막에 존재하는 α-9형의 아세틸콜린 수용체는 리간드의존성 Ca2 통로의 형태를 하고 있으므로11) Ca2 의 세포내 유입에 의한 K 통 로의 조절 가능성도 있다. 그러나 아직까지 Deiters 세포의 세포막 이온통로, 특히 K 통로에 대한 연구 보고는 미미한 Fig. 1. An isolated Deiters cell from guinea pig cochlea. Long phalangeal process shows the unique feature of Deiters cell (arrow). 551
- 552 Fig. 2. Activated current traces A and curve B recorded from Deiters cells. This current shows rapid activating outward rectifying current. - Korean J Otolaryngol 2002;45:550-6
Fig. 3. Calcium-independence of this outward current. Normal current A is not different from the current in Ca 2 -free normal Tyrode solution B. curves are also similar C. N/Tnormal Tyrode solution. Fig. 5. curve of different concentration of K o A and the fitting curve of the change of the reversal potential Fig. 4. Steady state inactivation and activation curves. The B. A External solution with higher K concentration shows activation curve is fitted by double Boltsmann equation suggesting the presence of more than two channels. Vhhalf of the measurement is 92.4 of the theoretical curve. Vrev more positive reversal potential. B The slope of the curve activation or inactivation voltage, Kslope factor. reversal potential. 553
Fig. 7. Ion selectivity of the outward rectifier current. K channel is also permeable to Cs and Na in its positive step pulses. Fig. 6. A Reactivation curve after activation from 120 mv to 60 mv, B the reactivation current after 80 mv prepulse of activation and C the relationship of the reactivation current and the delay after prepulse. Time constant Tau is the half reactivation time after activation. 554 Fig. 8. The effect of gadolinium. A The outward current partially inhibited by 100 M of gadolinium. B The curve of gadolinium-inhibited current. The steady state current was partially about 20 blocked by gadolinium. N/T normal Tyrode solution, Gdgadolinium. Korean J Otolaryngol 2002;45:550-6
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