Department of General and Environmental Physiology, University of Bari-Aldo Moro, Bari, Italy.
PLoS One. 2011 Apr 28;6(4):e19225. doi: 10.1371/journal.pone.0019225.
Aquaporin-4 (AQP4) is a water channel expressed at the sarcolemma of fast-twitch skeletal muscle fibers, whose expression is altered in several forms of muscular dystrophies. However, little is known concerning the physiological role of AQP4 in skeletal muscle and its functional and structural interaction with skeletal muscle proteome. Using AQP4-null mice, we analyzed the effect of the absence of AQP4 on the morphology and protein composition of sarcolemma as well as on the whole skeletal muscle proteome. Immunofluorescence analysis showed that the absence of AQP4 did not perturb the expression and cellular localization of the dystrophin-glycoprotein complex proteins, aside from those belonging to the extracellular matrix, and no alteration was found in sarcolemma integrity by dye extravasation assay. With the use of a 2DE-approach (BN/SDS-PAGE), protein maps revealed that in quadriceps, out of 300 Coomassie-blue detected and matched spots, 19 proteins exhibited changed expression in AQP4(-/-) compared to WT mice. In particular, comparison of the protein profiles revealed 12 up- and 7 down-regulated protein spots in AQP4-/- muscle. Protein identification by MS revealed that the perturbed expression pattern belongs to proteins involved in energy metabolism (i.e. GAPDH, creatine kinase), as well as in Ca(2+) handling (i.e. parvalbumin, SERCA1). Western blot analysis, performed on some significantly changed proteins, validated the 2D results. Together these findings suggest AQP4 as a novel determinant in the regulation of skeletal muscle metabolism and better define the role of this water channel in skeletal muscle physiology.
水通道蛋白-4（AQP4）是一种表达在快收缩骨骼肌纤维肌膜上的水通道，其表达在几种形式的肌肉营养不良中发生改变。然而，关于 AQP4 在骨骼肌中的生理作用及其与骨骼肌蛋白质组的功能和结构相互作用，人们知之甚少。使用 AQP4 基因敲除小鼠，我们分析了 AQP4 缺失对肌膜形态和蛋白质组成以及整个骨骼肌蛋白质组的影响。免疫荧光分析表明，AQP4 的缺失并没有扰乱肌营养不良糖蛋白复合物蛋白的表达和细胞定位，除了那些属于细胞外基质的蛋白，染料渗出试验也没有发现肌膜完整性的改变。通过 2-DE 方法（BN/SDS-PAGE），蛋白质图谱显示，在股四头肌中，在 300 个考马斯亮蓝检测到和匹配的斑点中，有 19 种蛋白质在 AQP4(-/-)小鼠中与 WT 小鼠相比表达发生了变化。特别是，蛋白质图谱的比较显示，AQP4(-/-)肌肉中有 12 个上调和 7 个下调的蛋白质斑点。通过 MS 鉴定的蛋白质表明，受干扰的表达模式属于涉及能量代谢的蛋白质（即 GAPDH、肌酸激酶），以及涉及 Ca(2+) 处理的蛋白质（即 parvalbumin、SERCA1）。对一些明显变化的蛋白质进行的 Western blot 分析验证了 2D 结果。这些发现共同表明 AQP4 是调节骨骼肌代谢的一个新的决定因素，并更好地定义了这种水通道在骨骼肌生理学中的作用。
