We investigated whether insulin-like growth factor-1 (IGF-1), an endogenous potent activator of skeletal muscle proliferation and differentiation, enhances L-type Ca2+ channel gene expression resulting in increased functional voltage sensors in single skeletal muscle cells. 2. Charge movement and inward Ca2+ current were recorded in primary cultured rat myoballs using the whole-cell configuration of the patch-clamp technique. Ca2+ current and maximum charge movement (Qmax) were potentiated in cells treated with IGF-1 without significant changes in their voltage dependence. Peak Ca2+ current in control and IGF-1-treated cells was -7.8 +/- 0.44 and -10. 5 +/- 0.37 pA pF-1, respectively (P < 0.01), whilst Qmax was 12.9 +/- 0.4 and 22.0 +/- 0.3 nC microF-1, respectively (P < 0.01). 3. The number of L-type Ca2+ channels was found to increase in the same preparation. The maximum binding capacity (Bmax) of the high-affinity radioligand [3H]PN200-110 in control and IGF-1-treated cells was 1.21 +/- 0.25 and 3.15 +/- 0.5 pmol (mg protein)-1, respectively (P < 0.01). No significant change in the dissociation constant for [3H]PN200-110 was found. 4. Antisense RNA amplification showed a significant increase in the level of mRNA encoding the L-type Ca2+ channel alpha1-subunit in IGF-1-treated cells. 5. This study demonstrates that IGF-1 regulates charge movement and the level of L-type Ca2+ channel alpha1-subunits through activation of gene expression in skeletal muscle cells.
摘要
我们研究了胰岛素样生长因子-1(IGF-1),一种骨骼肌增殖和分化的内源性强效激活剂,是否能增强L型Ca2+通道基因表达,从而导致单个骨骼肌细胞中功能性电压传感器增加。2. 使用膜片钳技术的全细胞配置,在原代培养的大鼠肌球中记录电荷移动和内向Ca2+电流。用IGF-1处理的细胞中Ca2+电流和最大电荷移动(Qmax)增强,其电压依赖性无显著变化。对照细胞和IGF-1处理细胞中的峰值Ca2+电流分别为-7.8±0.44和-10.5±0.37 pA pF-1(P<0.01),而Qmax分别为12.9±0.4和22.0±0.3 nC μF-1(P<0.01)。3. 在同一制备物中发现L型Ca2+通道数量增加。对照细胞和IGF-1处理细胞中高亲和力放射性配体[3H]PN200-110的最大结合容量(Bmax)分别为1.21±0.25和3.15±0.5 pmol(mg蛋白)-1(P<0.01)。未发现[3H]PN200-110的解离常数有显著变化。4. 反义RNA扩增显示,IGF-1处理细胞中编码L型Ca2+通道α1亚基的mRNA水平显著增加。5. 本研究表明,IGF-1通过激活骨骼肌细胞中的基因表达来调节电荷移动和L型Ca2+通道α1亚基的水平。