Department of Molecular Physiology and Biophysics, Vanderbilt University, 7425B MRB IV, Nashville, TN, USA.
J Physiol. 2020 Nov;598(21):4887-4905. doi: 10.1113/JP279941. Epub 2020 Sep 1.
Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how β-cell TSPAN-7 regulates Ca handling and hormone secretion. We find that TSPAN-7 reduces β-cell glucose-stimulated Ca entry, slows Ca oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls β-cell function through a direct interaction with L-type voltage-dependent Ca channels (Ca 1.2 and Ca 1.3), which reduces channel Ca conductance. TSPAN-7 slows activation of Ca 1.2 and accelerates recovery from voltage-dependent inactivation; TSPAN-7 also slows Ca 1.3 inactivation kinetics. These findings strongly implicate TSPAN-7 as a key regulator in determining the set-point of glucose-stimulated Ca influx and insulin secretion.
Glucose-stimulated insulin secretion (GSIS) is regulated by calcium (Ca ) entry into pancreatic β-cells through voltage-dependent Ca (Ca ) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca handling, and thus they may also modulate GSIS. TSPAN-7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN-7 is highly expressed in β- and α-cells; however, the function of islet TSPAN-7 has not been determined. Here, we show that TSPAN-7 knockdown (KD) increases glucose-stimulated Ca influx into mouse and human β-cells. Additionally, mouse β-cell Ca oscillation frequency was accelerated by TSPAN-7 KD. Because TSPAN-7 KD also enhanced Ca entry when membrane potential was clamped with depolarization, the effect of TSPAN-7 on Ca channel activity was examined. TSPAN-7 KD enhanced L-type Ca currents in mouse and human β-cells. Conversely, heterologous expression of TSPAN-7 with Ca 1.2 and Ca 1.3 L-type Ca channels decreased Ca currents and reduced Ca influx through both channels. This was presumably the result of a direct interaction of TSPAN-7 and L-type Ca channels because TSPAN-7 coimmunoprecipitated with both Ca 1.2 and Ca 1.3 from primary human β-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human β-cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN-7 modulation of β-cell L-type Ca channels is a key determinant of β-cell glucose-stimulated Ca entry and thus the set-point of GSIS.
四跨膜蛋白（TSPAN）调节许多生物过程，包括细胞内钙（Ca ）处理。TSPAN-7 在胰岛细胞中丰富，但胰岛 TSPAN-7 的功能尚未确定。在这里，我们描述了β细胞 TSPAN-7 如何调节 Ca 处理和激素分泌。我们发现 TSPAN-7 降低了β细胞葡萄糖刺激的 Ca 内流，降低了 Ca 振荡频率，并降低了葡萄糖刺激的胰岛素分泌。TSPAN-7 通过与 L 型电压依赖性 Ca 通道（Ca 1.2 和 Ca 1.3）的直接相互作用来控制β细胞功能，从而降低通道 Ca 电导。TSPAN-7 减慢 Ca 1.2 的激活并加速从电压依赖性失活中恢复；TSPAN-7 还减慢 Ca 1.3 的失活动力学。这些发现强烈表明 TSPAN-7 是决定葡萄糖刺激的 Ca 流入和胰岛素分泌设定点的关键调节剂。
葡萄糖刺激的胰岛素分泌（GSIS）受胰腺β细胞通过电压依赖性 Ca（Ca）通道进入 Ca 的调节。四跨膜蛋白（TSPAN）跨膜蛋白控制 Ca 处理，因此它们也可能调节 GSIS。TSPAN-7 是胰岛中最丰富的 TSPAN，对小鼠和人类胰腺切片的免疫染色显示 TSPAN-7 在β-和α-细胞中高度表达；然而，胰岛 TSPAN-7 的功能尚未确定。在这里，我们表明 TSPAN-7 敲低（KD）增加了小鼠和人类β细胞中葡萄糖刺激的 Ca 内流。此外，TSPAN-7 KD 还加速了小鼠β细胞的 Ca 振荡频率。因为 TSPAN-7 KD 在用去极化夹钳膜电位时也增强了 Ca 进入，所以检查了 TSPAN-7 对 Ca 通道活性的影响。TSPAN-7 KD 增强了小鼠和人类β细胞中的 L 型 Ca 电流。相反，L 型 Ca 通道的 TSPAN-7 异源表达与 Ca 1.2 和 Ca 1.3 降低了 Ca 电流并减少了两种通道的 Ca 流入。这可能是由于 TSPAN-7 与 L 型 Ca 通道的直接相互作用，因为 TSPAN-7 从原代人β细胞和异源表达系统中与 Ca 1.2 和 Ca 1.3 共免疫沉淀。最后，人β细胞中的 TSPAN-7 KD 增加了基础（5.6 mM 葡萄糖）和刺激（45 mM KCl + 14 mM 葡萄糖）胰岛素分泌。这些发现强烈表明，TSPAN-7 对β细胞 L 型 Ca 通道的调节是决定β细胞葡萄糖刺激的 Ca 内流和因此 GSIS 设定点的关键因素。
