ION CHANNELS IN VOLUME REGULATION BY NEUROBLASTOMA CELLS

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，液体输送上皮细胞， 外周血细胞，甚至神经胶质细胞和神经元最初都会肿胀，但 然后，在几分钟内，收缩到接近其静止体积。 调节性容量减少(RVD)通常伴随着被动的 细胞内K+和包括Cl-在内的阴离子的损失。 最近，利用细胞附着式膜片钳记录，我们研究了 克隆性N1E115细胞肿胀和RVD过程中的离子通道活性 神经母细胞瘤细胞。对拉伸敏感的、非选择性的活动 阳离子通道(C+(SA))在渗透性发作后不久升高 诱导细胞肿胀。不久之后，并大致与 RVD的发生，两种电压依赖的自发通道 在静息电位下打开：(1)延迟整流型K+通道， (2)大电导阴离子通道。我们假设C++ (Sa)通道可以是音量“传感器”机构，而电压- 依赖的钾离子和阴离子通道可能是钾盐退出途径。 房租和房租。 我们现在建议通过结合视频显微镜来检验后一种假设 使用(A)单通道记录和(B) 全细胞记录的穿孔贴片变体以检查 细胞肿胀和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，在 离子通道阻滞剂的存在和不存在，我们将向 对于给定的频道要有选择性。我们将集中精力确定(A)何时 以及每种离子通道类型在RVD过程中打开多少个；(B)什么力量 和/或细胞内信使实际上控制每个通道，以及 哪些离子在打开时渗入每个通道；及(C)净值 在RVD期间通过这些通道的离子外流占了很大一部分或全部 从细胞中流失的盐分。 这些结果可能有助于阐明脑细胞用来 低钠血症、脑缺血、神经毒性损伤时的限制肿胀 兴奋性递质，甚至细胞生长。

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.