Gerencser George A, Robbins Frank, Zhang Jianliang, Ahearn Gregory A
Department of Physiology and Functional Genomics, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
J Exp Biol. 2004 Feb;207(Pt 4):571-8. doi: 10.1242/jeb.00495.
The transport of [14C]oxalate (Ox2-) by epithelial brush-border membrane vesicles (BBMV) of lobster (Homarus americanus) hepatopancreas, formed by a magnesium precipitation technique, was stimulated by an outward Cl- gradient (in > out). By contrast, Ox2- uptake was not enhanced by an inward Na+ or K+ transmembrane gradient. Generation of an inside-positive membrane potential by K+ in the presence of valinomycin stimulated Ox2-/Cl- exchange, while an inside-negative membrane potential generated by K+ efflux in the presence of valinomycin inhibited this process. Neither Ox2-/Ox2- nor Ox2-/SO4(2-) transport exchange were affected by alterations of transmembrane potential. An inwardly directed proton gradient, or the presence of low bilateral pH, enhanced Ox2-/Cl- exchange, yet the H+ gradient alone could not stimulate Ox2) uptake in Cl(-)-equilibrated BBMV or in vesicles lacking internal Cl-. The stilbenes 4-acetamido-4'-isothiocyanotostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) strongly inhibited Ox2-/Cl- exchange. Oxalate influx occurred by a combination of carrier-mediated transfer, exhibiting Michaelis-Menten kinetics, and nonsaturable 'apparent diffusion'. Apparent kinetic constants for Ox2-/Cl- exchange were Kt = 0.20 mmol l(-1) and Jmax = 1.03 nmol l(-1) mg(-1) protein 7 s(-1). 36Cl- influx into oxalate-loaded BBMV was stimulated by an inside-negative transmembrane potential compared with short-circuited vesicles. These results suggest that Ox2-/Cl- exchange in crustacean hepatopancreatic BBMV occurred by an electrogenic carrier mechanism exhibiting a 1:1 flux ratio that was modulated by an external proton-sensitive regulatory site.
通过镁沉淀技术形成的美国龙虾（美洲螯龙虾）肝胰腺上皮刷状缘膜囊泡（BBMV）对[14C]草酸盐（Ox2-）的转运，受到外向Cl-梯度（胞内＞胞外）的刺激。相比之下，内向Na+或K+跨膜梯度不会增强Ox2-的摄取。在缬氨霉素存在下，K+产生的膜内正电位刺激了Ox2-/Cl-交换，而在缬氨霉素存在下，K+外流产生的膜内负电位则抑制了这一过程。跨膜电位的改变对Ox2-/Ox2-或Ox2-/SO4(2-)转运交换均无影响。内向质子梯度或双侧低pH的存在增强了Ox2-/Cl-交换，但单独的H+梯度无法刺激处于Cl-平衡的BBMV或缺乏内部Cl-的囊泡对Ox2-的摄取。芪类化合物4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸（SITS）和4,4'-二异硫氰基-2,2'-二磺酸芪（DIDS）强烈抑制Ox2-/Cl-交换。草酸盐内流是由载体介导的转运（呈现米氏动力学）和不饱和的“表观扩散”共同作用的结果。Ox2-/Cl-交换的表观动力学常数为Kt = 0.20 mmol l(-1)，Jmax = 1.03 nmol l(-1) mg(-1)蛋白7 s(-1)。与短路囊泡相比，膜内负跨膜电位刺激了36Cl-流入草酸盐负载的BBMV。这些结果表明，甲壳类动物肝胰腺BBMV中的Ox2-/Cl-交换是通过一种电生载体机制发生的，该机制表现出1:1的通量比，并受外部质子敏感调节位点的调控。
