Expression of the Na/P<Subscript>i</Subscript>-cotransporter Type IIb in Sf9 Cells: Functional Characterization and Purification

In mammals the type IIb Na/P_i-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/P_i-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/P_i-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of ~90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K _ms for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/P_i-cotransporter can be purified by Ni-NTA chromatography.     

Electrogenic Kinetics of a Mammalian Intestinal Type IIb Na+/Pi Cotransporter

The kinetics of a type IIb Na(+)-coupled inorganic phosphate (Pi) cotransporter (NaPi-IIb) cloned from mouse small intestine were studied using the two-electrode voltage clamp applied to Xenopus oocytes. In the steady state, mouse NaPi-IIb showed a curvilinear I-V relationship, with rate-limiting behavior only for depolarizing potentials. The Pi dose dependence was Michaelian, with an apparent affinity constant for Pi (Km(pi)) of 10 +/- 1 microM: at -60 mV. Unlike for rat NaPi-IIa, (Km(pi)) increased with membrane hyperpolarization, as reported for human NaPi-IIa, flounder NaPi-IIb and zebrafish NaPi-IIb2. The apparent affinity constant for Na(+) (Km(na)) was 23 +/- 1 mM: at -60 mV, and the Na(+) activation was cooperative with a Hill coefficient of approximately 2. Pre-steady-state currents were documented in the absence of Pi and showed a strong dependence on external Na(+). The hyperpolarizing shift of the charge distribution midpoint potential was 65 mV/log[Na]. Approximately half the moveable charge was attributable to the empty carrier. A comparison of the voltage dependence of steady-state Pi-induced current and pre-steady-state charge movement indicated that for -120 mV NaPi-IIb steady-state activity, independent of the titration of mono- and divalent Pi, and immobilized pre-steady-state charge movements associated with the first Na(+) binding step.     

急性一氧化碳中毒大鼠海马组织NaPi-IIb 蛋白表达的研究

目的:研究急性一氧化碳中毒后大鼠海马组织NaPi-IIb蛋白的表达情况。方法:选取雄性Sprague-Dawley(SD)大鼠48只,随机分为正常对照组(NC组)14只、缺氧组(IB组)17只、急性CO中毒组(CO-P组)17只。利用简易密闭容器建立缺氧模型;利用多次追加腹腔注射CO气体染毒的方法建立急性一氧化碳中毒模型。观察各组大鼠在建模过程中呼吸、活动改变情况。大鼠于建模24 h-26 h后取脑组织制作石蜡切片,选取海马组织平面利用免疫组织化学染色(IHC)观察组织形态及海马组织中NaPi-IIb蛋白的表达情况。同时,分离各组大鼠海马组织,利用蛋白质免疫印迹技术(Western Blot,WB)检测海马组织中NaPi-IIb蛋白的表达情况。结果:IHC、WB结果表明海马组织中NaPi-IIb蛋白的表达在CO-P组中明显高于NC组、IB组(P0.05)。NC组、IB组比较,NaPi-IIb蛋白的表达无明显差异(P0.05)。结论:在本研究中,CO暴露后,大鼠海马组织NaPi-IIb蛋白表达的增高与CO相关。NaPi-IIb蛋白可能成为CO中毒稳定、可靠的诊断标志物。