Characterization of Na+/Ca2+ Exchanger Isoforms in Rat Lens

Abstract

Purpose:: Although Ca²⁺ homeostasis is critical to lens transparency, the molecular mechanism of Ca²⁺ regulation remains unclear. To maintain intracellular Ca²⁺ concentration at the physiological level, the lens must export Ca²⁺ continuously. Na⁺/Ca²⁺ exchanger (NCX) is one of the essential regulators of Ca²⁺ homeostasis in many cells. NCX catalyzed reversible exchange of Na⁺ for Ca²⁺ across the plasma membrane. Transport activity of NCX is known to be influenced by variety of factors, including hormones and growth factor, intracellular Na⁺ and Ca²⁺ concentrations, membrane potential, cytoplasmic ATP, and protein and lipid phosphorylation. Recently, it was reported that selective inhibitors of the reverse mode of NCX1 are effective drugs against the lens transparency. To reveal the role of NCX in the control of lens Ca²⁺ homeostasis, we characterized NCX isoform expressed in rat lens.

Methods:: The lens from neonatal (post natal day 5, 7 and 9) and adult (120 days) Wistar rats were homogenized by Hiscotron (NITI-ON) in the lysis buffer containing 20mM HEPES (pH 7.4), 150mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 2µg/ml leupeptin, 1µg/ml aprotinin, 200µM phenylmethylsulfonyl fluoride, and 200mM benzamidine hydrochroride. The lysates were subjected to centrifugation at 15,000g for 30 min, and resultant supernatants were subjected to SDS-PAGE on 8.5% gel and then to immunoblotting with anti-NCX1, 2, and 3 antibody. The immunoblot was visualized using an enhanced chemiluminescence detection system (ATTO).

Results:: Anti-NCX1 antibody recognized the molecule of 120KDa, which is mature NCX1, in both neonatal and adult rat lens, while Anti-NCX2 and 3 antibodies did not detect any band.

Conclusions:: In rat lens, including developmental period, NCX1 acts a role as a Ca²⁺ extrusion system to keep the intracellular Ca²⁺ concentration. To establish the causal relationship between NCX activity and lens transparency, we are currently trying a characterization and biochemical analysis of NCX1 using the lens of experimental rat cataract models.

Keywords: ion transporters