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: 3414438
• 负责人: STANLEY MISLER
• 金额: $ 10.07万
• 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/3414438
• 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。 什么时候 置于低渗浴液中，输送上皮细胞的液体， 外周血细胞甚至神经胶质细胞和神经元最初都会肿胀，但是 然后，在几分钟内，缩小到接近静止状态的体积。 调节量减少（RVD）通常伴随着被动 细胞内 K+ 和阴离子（包括 Cl-）的损失。 最近，使用细胞贴附膜片钳记录，我们研究了 克隆 N1E115 细胞肿胀和 RVD 期间的离子通道活性 神经母细胞瘤细胞。 拉伸敏感、非选择性的活动 渗透压开始后不久，阳离子通道 (C+(SA)) 增加 引起细胞肿胀。 此后不久，大致与 RVD 发生时，两种类型的电压依赖性通道自发出现 在静息电位处打开： (1) 延迟整流器型 K+ 通道， (2)大电导阴离子通道。 我们假设 C+ (SA)通道可能是音量“传感器”机制，而电压- 依赖的 K+ 和阴离子通道可能是钾盐排出途径 RVD。 我们现在建议通过结合视频显微镜来检验后一个假设 使用 (a) 单通道记录和 (b) 细胞大小成像 全细胞记录的穿孔贴片变体，用于检查变化 细胞膨胀和 RVD 过程中的膜电流、电压和电阻 神经母细胞瘤在各种渗透压扰动下，并且在 我们将证明是否存在离子通道阻滞剂 对给定的渠道有选择性。 我们将重点确定 (a) 何时 以及 RVD 期间每种离子通道类型打开多少个； (b) 什么力量 和/或细胞内信使实际上门控每个通道，以及 打开时哪些离子会渗透到每个通道； (c) 净额是否 RVD 期间通过这些通道的离子流出占大部分或全部 细胞中盐的损失。 这些结果可能有助于阐明脑细胞使用的机制 限制低钠血症、缺血、神经毒物损伤期间的肿胀 兴奋性递质，甚至细胞生长。