ION CHANNELS IN VOLUME REGULATION BY NEUROBLASTOMA CELLS

神经母细胞瘤细胞体积调节中的离子通道

• 批准号: 3414439
• 负责人: STANLEY MISLER
• 金额: $ 8.94万
• 依托单位: JEWISH HOSPITAL OF SAINT LOUIS
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3414439
• 关键词: biological fluid transport; brain cell; calcium channel; cell osmotic pressure; cell water; electrophysiology; hyponatremia; ion transport; membrane channels; microscopy; neoplastic cell culture for noncancer research; neuroblastoma; potassium channel; second messengers; tissue /cell culture; video recording system; voltage /patch clamp; voltage gated channel

Volume regulation is a feature common to many vertebrate cells. When placed in hypotonic bathing solution, fluid transporting epithelial cells, peripheral blood cells and even glia and neurons initially swell, but then, over several minutes, shrink back to near their resting volumes. The regulatory volume decrease (RVD) is usually accompanied by the passive loss of intracellular K+ and anions including Cl-. Recently, using cell-attached patch clamp recording, we have investigated ion channel activity during cell swelling and RVD in clonal N1E115 neuroblastoma cells. The activity of a stretch-sensitive, non-selective cation channel (C+(SA)) increases shortly after the onset of osmotically induced cell swelling. Shortly thereafter, and roughly coincident with the onset of RVD, two types of voltage-dependent channels spontaneously open at the resting potential: (1) a delayed-rectifier type K+ channel, and (2) large conductance anion channel. We have hypothesized that the C+ (SA) channel may be a volume "sensor" mechanism, while the voltage- dependent K+ and anion channels may be potassium salt exit pathways during RVD. We now propose to test the latter hypothesis by combining videomicroscopic imaging of cell size with (a) single-channel recording and (b) the perforated patch variant of whole-cell recording to examine changes in membrane current, voltage and resistance during cell swelling and RVD in neuroblastoma under a variety of osmotic perturbations, and in the presence and absence of ion channel blockers which we shall demonstrate to be selective for a given channel. We shall focus on ascertaining (a) when and how many each ion channel type opens during RVD; (b) what forces and/or intracellular messengers actually gate each channel, as well as which ions permeate each channel when open; and (c) whether the net sum ion efflux through these channel during the RVD accounts for much or all of the salt loss from the cell. These results may help to elucidate the mechanism used by brain cells to limit swelling during hyponatremia, ischemia, insult by neurotoxic excitatory transmitters, and even cell growth.

体积调节是许多脊椎动物细胞的共同特征。 当 放置在低渗浴溶液中，液体运输上皮细胞， 外周血细胞甚至神经胶质和神经元最初会肿胀， 然后，几分钟后，收缩到接近它们的静止体积。 调节性血容量减少（RVD）通常伴随着被动的 细胞内K+和阴离子（包括Cl-）的损失。 最近，我们利用细胞贴附式膜片钳记录， 克隆N1 E115细胞肿胀和RVD过程中离子通道活性 神经母细胞瘤细胞 一种对拉伸敏感的非选择性 阳离子通道（C+（SA））在急性心肌梗死发作后不久增加， 诱导细胞肿胀。 此后不久，大致与 RVD发作时，两种电压依赖性通道自发 在静息电位下开放：（1）延迟整流型K+通道， （2）大电导阴离子通道。 我们假设C++ (SA)通道可以是体积“传感器”机制，而电压- 依赖性K+和阴离子通道可能是钾盐的出口途径， RVD. 我们现在建议通过结合视频显微镜来测试后一种假设。 用（a）单通道记录和（B） 全细胞记录的穿孔贴片变体，以检查 细胞膨胀过程中的膜电流、电压和电阻以及RVD 神经母细胞瘤下的各种渗透扰动，并在 存在和不存在离子通道阻滞剂，我们将证明， 对于给定的频道是有选择性的。 我们将集中精力确定（a）何时 以及在RVD期间每种离子通道类型打开多少;（B）什么力 和/或细胞内信使实际上门控每个通道，以及 当打开时，哪些离子渗透每个通道;以及（c）净总和是否 在RVD期间通过这些通道的离子流出占大部分或全部 细胞中盐分流失的最大程度 这些结果可能有助于阐明脑细胞用于 在低钠血症、缺血、神经毒性损伤期间限制肿胀 兴奋性递质甚至细胞生长