Herring T L, Slotin I M, Baltz J M, Morris C E
Department of Biology, University of Ottawa, Ontario, Canada.
Am J Physiol. 1998 Jan;274(1):C272-81. doi: 10.1152/ajpcell.1998.274.1.C272.
Neurons are mechanically robust. During prolonged swelling, molluscan neurons can triple their apparent membrane area. They gain surface area and capacitance independent of extracellular Ca concentration ([Ca]e), but it is unknown if an increase in intracellular Ca concentration ([Ca]i) is necessary. If Ca for stimulating exocytosis is unnecessary, it is possible that swelling-induced membrane tension changes directly trigger surface area readjustments. If, however, Ca-mediated but not tension-mediated membrane recruitment is responsible for surface area increases, swelling neurons should sustain elevated levels of [Ca]i. The purpose of this investigation is to determine if the [Ca]i in swelling neurons attains levels high enough to promote exocytosis and if any such increase is required. Lymnaea neurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 microM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min) were done at either a standard [Ca]e or very low [Ca]e level (0.9 mM or 0.13 microM, respectively). In neither case did the [Ca]i increase to levels that drive exocytosis. We also monitored osmomechanically driven membrane dynamics [swelling, then formation and reversal of vacuole-like dilations (VLDs)] with the [Ca]i clamped below 40 nM via 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]i did not change with swelling, and VLD behavior was unaffected, consistent with tension-driven, [Ca]i-independent surface area adjustments. In addition, neurons with [Ca]i clamped at 0.1 microM via an ionophore could produce VLDs. We conclude that, under mechanical stress, neuronal membranes are compliant by virtue of surface area regulatory adjustments that operate independent of [Ca]i. The findings support the hypothesis that plasma membrane area is regulated in part by membrane tension.
神经元在机械性能上具有很强的韧性。在长时间肿胀过程中，软体动物的神经元其表观膜面积可增加两倍。它们增加表面积和电容与细胞外钙浓度（[Ca]e）无关，但细胞内钙浓度（[Ca]i）的升高是否必要尚不清楚。如果刺激胞吐作用的钙并非必需，那么肿胀诱导的膜张力变化可能直接触发表面积的重新调整。然而，如果是钙介导而非张力介导的膜募集导致表面积增加，那么肿胀的神经元应该维持较高水平的[Ca]i。本研究的目的是确定肿胀神经元中的[Ca]i是否达到足以促进胞吐作用的水平，以及是否需要任何此类增加。将椎实螺神经元用钙浓度指示剂fura 2进行负载。使用4-溴-A-23187和钙-乙二醇双（β-氨基乙基醚）-N,N,N',N'-四乙酸（EGTA）原位进行校准，游离钙浓度范围为0至5微摩尔。在标准[Ca]e水平或非常低的[Ca]e水平（分别为0.9毫摩尔或0.13微摩尔）下进行肿胀扰动（将培养基渗透压降低至25%，持续5分钟）。在这两种情况下，[Ca]i均未升高至驱动胞吐作用的水平。我们还通过1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）将[Ca]i钳制在40纳摩尔以下，监测渗透机械驱动下膜的动态变化[肿胀，然后形成和逆转液泡样扩张（VLDs）]。[Ca]i并未随肿胀而变化，VLD行为也未受影响，这与张力驱动、与[Ca]i无关的表面积调整一致。此外，通过离子载体将[Ca]i钳制在0.1微摩尔的神经元能够产生VLDs。我们得出结论，在机械应力作用下，神经元膜通过独立于[Ca]i的表面积调节调整而具有顺应性。这些发现支持了质膜面积部分受膜张力调节的假说。
