Olsen M L, Weaver A K, Ritch P S, Sontheimer H
Department of Neurobiology and Civitan International Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
J Neurosci Res. 2005 Jul 15;81(2):179-89. doi: 10.1002/jnr.20543.
Glioma cells show up-regulation and constitutive activation of erbB2, and its expression correlates positively with increased malignancy. A similar correlation has been demonstrated for the expression of gBK, a calcium-sensitive, large-conductance K(+) channel. We show here that glioma BK channels are a downstream target of erbB2/neuregulin signaling. Tyrphostin AG825 was able to disrupt the constituitive erbB2 activation in a dose-dependent manner, causing a 30-mV positive shift in gBK channel activation in cell-attached patches. Conversely, maximal stimulation of erbB2 with a recombinant neuregulin (NRG-1beta) caused a 12-mV shift in the opposite direction. RT-PCR studies reveal no change in the BK splice variants expressed in treated glioma cells. Furthermore, isolation of surface proteins through biotinylation did not show a change in gBK channel expression, and probing with phospho-specific antibodies showed no alteration in channel phosphorylation. However, fura-II Ca(2+) fluorescence imaging revealed a 35% decrease in the free intracellular Ca(2+) concentration after erbB2 inhibition and an increase in NRG-1beta-treated cells, suggesting that the observed changes most likely were due to alterations in Ca(2+). Consistent with this conclusion, neither tyrphostin AG825 nor NRG-1beta was able to modulate gBK channels under inside-out or whole-cell recording conditions when intracellular Ca(2+) was fixed. Thus, gBK channels are a downstream target for the abundantly expressed neuregulin-1 receptor erbB2 in glioma cells. However, unlike the case in other systems, this modulation appears to occur via changes in Ca(2+) without changes in channel expression or phosphorylation. The enhanced sensitivity of gBK channels in glioma cells to small, physiological Ca(2+) changes appears to be a prerequisite for this modulation.
胶质瘤细胞显示出erbB2的上调和组成性激活，其表达与恶性程度增加呈正相关。对于钙敏感的大电导钾通道gBK的表达也证明了类似的相关性。我们在此表明，胶质瘤BK通道是erbB2/神经调节蛋白信号传导的下游靶点。酪氨酸磷酸化抑制剂AG825能够以剂量依赖的方式破坏erbB2的组成性激活，导致细胞贴附膜片中gBK通道激活发生30 mV的正向偏移。相反，用重组神经调节蛋白（NRG-1β）对erbB2进行最大刺激会导致相反方向的12 mV偏移。RT-PCR研究显示，处理后的胶质瘤细胞中表达的BK剪接变体没有变化。此外，通过生物素化分离表面蛋白未显示gBK通道表达有变化，用磷酸化特异性抗体检测也未显示通道磷酸化有改变。然而，fura-II Ca(2+)荧光成像显示，erbB2抑制后细胞内游离Ca(2+)浓度降低35%，而NRG-1β处理的细胞中则升高，这表明观察到的变化很可能是由于[Ca(2+)]i的改变。与这一结论一致，当细胞内Ca(2+)固定时，酪氨酸磷酸化抑制剂AG825和NRG-1β在膜内向外或全细胞记录条件下均无法调节gBK通道。因此，gBK通道是胶质瘤细胞中大量表达的神经调节蛋白-1受体erbB2的下游靶点。然而，与其他系统不同的是，这种调节似乎是通过[Ca(2+)]i的变化而发生的，而通道表达或磷酸化没有变化。胶质瘤细胞中gBK通道对微小生理Ca(2+)变化的增强敏感性似乎是这种调节的前提条件。
