Peng J B, Chen X Z, Berger U V, Vassilev P M, Tsukaguchi H, Brown E M, Hediger M A
Membrane Biology Program, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1999 Aug 6;274(32):22739-46. doi: 10.1074/jbc.274.32.22739.
Calcium is a major component of the mineral phase of bone and serves as a key intracellular second messenger. Postnatally, all bodily calcium must be absorbed from the diet through the intestine. Here we report the properties of a calcium transport protein (CaT1) cloned from rat duodenum using an expression cloning strategy in Xenopus laevis oocytes, which likely plays a key role in the intestinal uptake of calcium. CaT1 shows homology (75% amino acid sequence identity) to the apical calcium channel ECaC recently cloned from vitamin D-responsive cells of rabbit kidney and is structurally related to the capsaicin receptor and the TRP family of ion channels. Based on Northern analysis of rat tissues, a 3-kilobase CaT1 transcript is present in rat duodenum, proximal jejunum, cecum, and colon, and a 6.5-kilobase transcript is present in brain, thymus, and adrenal gland. In situ hybridization revealed strong CaT1 mRNA expression in enterocytes of duodenum, proximal jejunum, and cecum. No signals were detected in kidney, heart, liver, lung, spleen, and skeletal muscle. When expressed in Xenopus oocytes, CaT1 mediates saturable Ca(2+) uptake with a Michaelis constant of 0.44 mM. Transport of Ca(2+) by CaT1 is electrogenic, voltage-dependent, and exhibits a charge/Ca(2+) uptake ratio close to 2:1, indicating that CaT1-mediated Ca(2+) influx is not coupled to other ions. CaT1 activity is pH-sensitive, exhibiting significant inhibition by low pH. CaT1 is also permeant to Sr(2+) and Ba(2+) (but not Mg(2+)), although the currents evoked by Sr(2+) and Ba(2+) are much smaller than those evoked by Ca(2+). The trivalent cations Gd(3+) and La(3+) and the divalent cations Cu(2+), Pb(2+), Cd(2+), Co(2+), and Ni(2+) (each at 100 microM) do not evoke currents themselves, but inhibit CaT1-mediated Ca(2+) transport. Fe(3+), Fe(2+), Mn(2+), and Zn(2+) have no significant effects at 100 microM on CaT1-mediated Ca(2+) transport. CaT1 mRNA levels are not responsive to 1,25-dihydroxyvitamin D(3) administration or to calcium deficiency. Our studies strongly suggest that CaT1 provides the principal mechanism for Ca(2+) entry into enterocytes as part of the transcellular pathway of calcium absorption in the intestine.
钙是骨骼矿质相的主要成分，也是关键的细胞内第二信使。出生后，身体所需的所有钙都必须通过肠道从饮食中吸收。在此，我们报告了利用非洲爪蟾卵母细胞中的表达克隆策略从大鼠十二指肠克隆出的一种钙转运蛋白（CaT1）的特性，该蛋白可能在肠道钙吸收中起关键作用。CaT1与最近从兔肾维生素D反应性细胞中克隆出的顶端钙通道ECaC具有同源性（氨基酸序列同一性为75%），并且在结构上与辣椒素受体和离子通道的TRP家族相关。基于对大鼠组织的Northern分析，在大鼠十二指肠、空肠近端、盲肠和结肠中存在一个3千碱基的CaT1转录本，在脑、胸腺和肾上腺中存在一个6.5千碱基的转录本。原位杂交显示在十二指肠、空肠近端和盲肠的肠细胞中有强烈的CaT1 mRNA表达。在肾、心、肝、肺、脾和骨骼肌中未检测到信号。当在非洲爪蟾卵母细胞中表达时，CaT1介导可饱和的Ca(2+)摄取，米氏常数为0.44 mM。CaT1介导的Ca(2+)转运是生电的、电压依赖性的，并且电荷/Ca(2+)摄取比接近2:1，这表明CaT1介导的Ca(2+)内流不与其他离子偶联。CaT1活性对pH敏感，在低pH时表现出显著抑制。CaT1对Sr(2+)和Ba(2+)（但对Mg(2+)不）也具有通透性，尽管Sr(2+)和Ba(2+)诱发的电流比Ca(2+)诱发的电流小得多。三价阳离子Gd(3+)和La(3+)以及二价阳离子Cu(2+)、Pb(2+)、Cd(2+)、Co(2+)和Ni(2+)（均为100 microM）本身不诱发电流，但抑制CaT1介导的Ca(2+)转运。100 microM的Fe(3+)、Fe(2+)、Mn(2+)和Zn(2+)对CaT1介导的Ca(2+)转运无显著影响。CaT1 mRNA水平对给予1,25 - 二羟维生素D(3)或钙缺乏无反应。我们的研究强烈表明，CaT1作为肠道钙吸收跨细胞途径的一部分，为Ca(进入肠细胞提供了主要机制。 2+)
