Weskamp M, Seidl W, Grissmer S
Department of Applied Physiology, University Ulm, 89081 Ulm, Germany.
J Membr Biol. 2000 Nov 1;178(1):11-20. doi: 10.1007/s002320010010.
The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, Ca(2+), in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in Ca(2+) from approximately 150 nm up to 1.3 microm. This increase in Ca(2+) was mainly due to an influx of extracellular Ca(2+) since removing of extracellular Ca(2+) reduced this increase to approximately 250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in Ca(2+) on membrane currents in these cells. An increase in Ca(2+) revealed two types of Ca(2+)-activated K(+) channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with Ca(2+) buffered to 100 nm or less. With patch-electrodes filled with solutions buffering Ca(2+) to 10 microm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from -160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than -40 mV from a holding potential of -80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in Ca(2+) elicited through hypotonic solutions. In combination with the volume-regulated Cl(-) conductance in the same cells this K(+) channel seems to play a vital role in volume regulation in human osteoblasts.
采用钙指示剂Fura-2研究低渗溶液对人成骨样细胞系细胞内钙浓度[Ca(2+)]i的影响。将细胞外溶液的张力降至50%会导致[Ca(2+)]i从约150 nM增加至1.3 μM。[Ca(2+)]i的这种增加主要是由于细胞外Ca(2+)的内流，因为去除细胞外Ca(2+)会使这种增加减少至约250 nM。细胞肿胀后，大多数细胞能够在800秒内将其体积调节至初始水平。膜片钳技术的全细胞记录模式也用于研究[Ca(2+)]i增加对这些细胞膜电流的影响。[Ca(2+)]i的增加揭示了两种类型的钙激活钾通道（K(Ca)通道）。当[Ca(2+)]i缓冲至100 nM或更低时，在+80 mV以下的电压下无法观察到通过这两种通道类型的电流。当膜片电极填充有将[Ca(2+)]i缓冲至10 μM的溶液时，可以很容易地观察到这两种通道类型。第一种类型的激活显然与电压无关，因为在从-160至+100 mV的整个使用电压范围内都可以观察到电流。此外，该电流也被蝎毒素（CTX）阻断。这些细胞中的第二种类型的K(Ca)通道可以在从-80 mV的保持电位去极化至比-40 mV更正的电位时被激活。这种类型被CTX和鬼笔环肽阻断。向细胞外溶液中添加鬼笔环肽会抑制调节性容积减小（RVD），但不能消除RVD。我们得出结论，人成骨细胞中存在两种类型的K(Ca)通道，一种是中等电导的K(Ca)通道和一种类大电导钙激活钾通道（MaxiK样K(Ca)通道）。MaxiK通道可能直接被激活，或者通过低渗溶液引起的[Ca(2+)]i增加而被激活。与同一细胞中受容积调节的Cl(-)电导相结合，这种K(+)通道似乎在人成骨细胞的容积调节中起着至关重要的作用。
