Hirsch J R, Loo D D, Wright E M
Department of Physiology, UCLA School of Medicine, Los Angeles, California 90095-1751, USA.
J Biol Chem. 1996 Jun 21;271(25):14740-6. doi: 10.1074/jbc.271.25.14740.
Cotransporters are proteins responsible for the accumulation of nutrients, neurotransmitters, and drugs in cells. As forskolin has been shown to stimulate intestinal Na+/glucose cotransport, we have used electrophysiological techniques to examine the role of protein kinases in regulating Na+/glucose cotransporters, SGLT1, expressed in Xenopus laevis oocytes. We monitored SGLT1 kinetics, the number of SGLT1 cotransporters in the plasma membrane, and plasma membrane area before and after activation of protein kinases. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and sn-1, 2-dioctanoylglycerol (DOG) were used as membrane permeable activators of protein kinases A (PKA) and C (PKC), respectively. In oocytes expressing rabbit SGLT1 8-Br-cAMP increased by 28 +/- 4% (n = 10), and DOG decreased by 51 +/- 5% (n = 13) the maximum rate of Na+/glucose cotransport. These reversible changes in the maximum transport rate occurred within minutes, and were accompanied by proportional changes in the number of cotransporters in the membrane and area of the plasma membrane. This suggests that protein kinases regulate rabbit SGLT1 activity by controlling the distribution of transporters between intracellular compartments and the plasma membrane, and that this occurs by exo- and endocytosis. Similar increases in maximum transport were obtained with activation of PKA in oocytes expressing rabbit, human, and rat SGLT1 isoforms, but with activation of PKC the response was isoform-dependent. PKC activation decreased the maximum rate of transport by rabbit and rat SGLT1, but increased transport by human SGLT1. We conclude that: (i) the regulation of SGLT1 expression in oocytes by protein kinases occurs mainly by regulated endo- and exocytosis; (ii) it is independent of consensus phosphorylation sites in the transporter; and (iii) the effect of a given kinase depends upon the actual sequence of the cotransporter expressed. These considerations may also apply to the regulation of other cotransporters by protein kinases in oocytes, cells, and tissues.
协同转运蛋白是负责细胞内营养物质、神经递质和药物积累的蛋白质。由于已证明福斯可林能刺激肠道钠/葡萄糖协同转运，我们运用电生理技术来研究蛋白激酶在调节非洲爪蟾卵母细胞中表达的钠/葡萄糖协同转运蛋白SGLT1中的作用。我们监测了蛋白激酶激活前后SGLT1的动力学、质膜中SGLT1协同转运蛋白的数量以及质膜面积。8-溴腺苷3',5'-环磷酸（8-Br-cAMP）和sn-1,2-二辛酰甘油（DOG）分别用作蛋白激酶A（PKA）和蛋白激酶C（PKC）的膜通透性激活剂。在表达兔SGLT1的卵母细胞中，8-Br-cAMP使钠/葡萄糖协同转运的最大速率增加了28±4%（n = 10），而DOG使其降低了51±5%（n = 13）。这些最大转运速率的可逆变化在数分钟内发生，并伴随着膜中协同转运蛋白数量和质膜面积的相应变化。这表明蛋白激酶通过控制转运蛋白在细胞内区室和质膜之间的分布来调节兔SGLT1的活性，并且这是通过胞吐作用和内吞作用发生的。在表达兔、人和大鼠SGLT1亚型的卵母细胞中，激活PKA可获得类似的最大转运增加，但激活PKC时反应具有亚型依赖性。PKC激活降低了兔和大鼠SGLT1的最大转运速率，但增加了人SGLT1的转运。我们得出以下结论：（i）蛋白激酶对卵母细胞中SGLT1表达的调节主要通过受调控的内吞作用和胞吐作用发生；（ii）它独立于转运蛋白中的共有磷酸化位点；（iii）给定激酶的作用取决于所表达的协同转运蛋白的实际序列。这些考虑因素也可能适用于卵母细胞、细胞和组织中蛋白激酶对其他协同转运蛋白的调节。
