Calcium Store-Operated Currents in Human Skin Cells
Calcium Store-Operated Currents in Human Skin Cells
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人体皮肤细胞中钙库控制的电流
DOI：
10.1016/j.bpj.2019.11.3070
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发表时间：
2020
影响因子：
3.4
通讯作者：
Manning D
中科院分区：
文献类型：
--
作者：
Manning D
角质形成细胞暴露于外表皮中增加的细胞外钙浓度[Ca 2+ o]触发分化以形成皮肤的保护性外层。增加的[Ca 2+ o]由细胞外Ca 2+敏感受体检测，其与IP 3产生偶联并导致内部Ca 2+储存的耗尽。随后的钙库操纵的钙内流（SOCE）通过影响数百个基因的转录控制来控制角质形成细胞的分化。在这里，我们调查的离子电流，有助于SOCE在角质形成细胞。逆转录PCR和免疫印迹表明，在转录和蛋白质水平的人角质形成细胞（HaCaT）细胞系中的STIM 1 -2，Orai 1 -3和多个TRP家族成员的表达。定量PCR显示Orai 1表达比Orai 2/3高5倍，STIM 1表达比STIM 2高18倍。为了使SOCE最大化，在将2 mM Ca 2+重新引入细胞外溶液之前，通过将细胞浸泡在含有2μM毒胡萝卜素的名义上无Ca 2+的溶液中来耗尽HaCaT Ca 2+储存。2 mMCa 2+ o再注入HaCaTs后，其全细胞电流呈现典型的双相性，由快速内向电流组成，在10-20 s内达到峰值62.6± 10.9pA/pF（n= 4），然后衰减至10.1± 1.7pA/pF（n= 4）。稳定期可能反映了维持的SOCE，因为由于毒胡萝卜素暴露，商店无法再填充。平台期被奥赖抑制剂GSK-7975 A部分抑制（1 µM;抑制77.6±13.7%; n= 4），尽管在平台期运行的电压斜坡产生了向外整流电流，与Orai介导的电流不一致。
Exposure of keratinocytes to increasing extracellular calcium concentrations [Ca 2+ o] in the outer epidermis triggers differentiation to form the protective outer layer of skin. Increasing [Ca 2+ o] is detected by the extracellular Ca 2+-sensing receptor which couples to IP 3 generation and leads to depletion of the internal Ca 2+ stores. Subsequent store-operated Ca 2+ entry (SOCE) controls keratinocyte differentiation through effects on the transcriptional control of hundreds of genes. Here we investigate the ionic currents that contribute to SOCE in keratinocytes.Reverse transcription-PCR and immunoblotting indicate expression of STIM1-2, Orai1-3 and multiple TRP family members at both transcript and protein level in a human keratinocyte (HaCaT) cell line. Quantitative PCR revealed Orai1 expression to be 5-fold higher than Orai2/3, and STIM1 expression 18-fold higher than STIM2. To maximise SOCE, HaCaT Ca 2+ stores were depleted by bathing cells in nominally Ca 2+-free solution containing 2µM thapsigargin before reintroducing 2mM Ca 2+ to the extracellular solution. Whole-cell currents recorded from HaCaTs in response to re-introduction of 2mM Ca 2+ o were typically biphasic and consisted of a ‘fast’inward current, which reached a peak magnitude of 62.6±10.9 pA/pF within 10-20 s (n= 4) before decaying to a sustained inward current plateau of 10.1±1.7 pA/pF (n= 4). The plateau likely reflects maintained SOCE since the stores are unable to refill due to thapsigarin exposure. The plateau was partially inhibited by the Orai inhibitor GSK-7975A (1 µM; inhibition 77.6±13.7%; n= 4), although voltage ramps run during the plateau phase generated an outwardly-rectifying current, inconsistent with Orai-mediated currents.