小鼠破骨细胞钠钙交换体亚型1的表达与功能

目的钠钙交换体(Sodium-Calcium Exchange,NCX)是钙离子排出细胞的重要途径。本研究探讨小鼠破骨细胞表达NCX1的不同剪切异构体及其钠钙交换功能。方法培养小鼠破骨细胞,纯化后提取总RNA样本,通过PCR克隆和DNA测序鉴定破骨细胞表达NCX1的具体剪切异构体种类。再将NCX1读码框架亚克隆至过表达质粒pIRESpuro,转染HEK293细胞后观察细胞内游离钙离子浓度的变化。结果在小鼠破骨细胞内克隆得到NCX1BD和NCX1ABD两个剪切异构体,其中NCX1ABD为新发现剪切异构体。将NCX1BD和NCX1ABD亚克隆至pIRESpuro后转染HEK293细胞,成功获得过表达细胞株。细胞加载钙离子荧光染色剂Furo-2/AM,抑制细胞膜纳钾泵并降低细胞外钠浓度后可以观察到NCX1反向模式导致的细胞内游离钙离子浓度的上升。但NCX1ABD导致的细胞内钙上升仅为NCX1BD的40%左右(t-检验,P0.001)。结论小鼠破骨细胞表达两种NCX1剪切异构体,其中NCX1ABD为新发现剪切异构体,可能具有较弱的钠钙交换功能。

Functional presence of sodium-calcium exchanger 1 variants in mouse osteoclasts

Objective Sodium-Calcium Exchange(NCX)is an important pathway of calcium efflux in mammal cells.This study is devoted to explore the expression and function of different splicing variants of NCX1 in mouse osteoclasts.Methods Total RNA was extracted from in vitro cultured mouse osteoclasts.The NCX1 transcripts were amplified by reverse transcription-polymerase chain reaction(RT-PCR).After cloning and DNA-sequencing,the splicing variants of NCX1 were identified.The open reading frames of the NCX1 variants were sub-cloned to vector pIRESpuro and transfected into HEK293 cells.The intracellular free calcium concentration was then monitored.Results Two splicing variants of NCX1 including NCX1BD and the newly-discovered NCX1ABD were identified in mouse osteoclasts.Their open reading frames were transfected and expressed in HEK293 cells.Cells expressing NCX1BD or NCX1ABD responded to the depletion of extracellular sodium by increased intracellular calcium.However,the calcium transient caused by NCX1ABD was only 40% of that caused by NCX1BD(t-test,P< 0.001).Conclusions Mouse osteoclasts express two NCX1 splicing variants NCX1BD and NCX1ABD.NCX1ABD is a newly-discovered sodium-calcium exchanger variant.