Ca2+ Homeostasis and Gap Junctional Coupling in Mouse Lenses with Genetically Altered Connexin Expression

Abstract

Abstract: : Purpose: Disturbance in [Ca²⁺] within the lens may be directly linked to cataracts. In the present study, we measured [Ca²⁺]_i at different depths in lenses from wild type, Cx46 for Cx50 knock-in (KI) and Cx46 knock-out (KO cataractous lens) mice to investigate Ca²⁺ homeostasis. Methods: All experiments were performed on freshly dissected mouse lenses. Since the lens absorbs light at the wavelengths of interest in a depth-dependent manner, [Ca²⁺]-calibration curves at different depths within the lens were generated by inserting a long sharp glass pipette, containing buffer solution with known free [Ca²⁺] plus Fura-2, into the center of the lens, then measuring the ratio of fluorescence at 360:380 nm at different [Ca²⁺]. However, the K_0.5 of the buffer solution differs from that of cytoplasm, so we incubated vesicles (which budded off of frog lens fibers) with known [Ca²⁺] plus Fura-2 AM and the Ca-ionophore A23187, to obtain the in vivo K_0.5, then shifted the buffer calibration curves accordingly. To measure [Ca²⁺]_i within the lens, we injected Fura-2 into the fiber cells at different depths, then recorded the ratio of fluorescence, and these data were converted into [Ca²⁺] with the calibration curve from the appropriate depth. Results: In wild type lenses (average diameter 2.05±0.05 mm), [Ca²⁺]_i increased continuously with distance from the lens surface. In surface fiber cells it was approximately 400 nM whereas in cells at the lens center it was about 700 nM. This gradient is consistent with a circulation of Ca²⁺ into the lens along extracellular spaces, crossing membranes into fiber cells, then diffusing from cell to cell through gap junctions to the surface where it is actively transported out of the lens. The mature fiber cell gap junctional conductance was 0.49 S/cm² of cell to cell contact in wild type lenses. A much smaller [Ca²⁺]_i-gradient was present in KI lenses, which had a concentration of 300 nM in surface cells and 450 nM in central cells. The KI lenses also had a much higher gap junctional coupling conductance of 1.30 S/cm², consistent with the circulation hypothesis. The KO lenses lacked gap junctional coupling of mature fibers, where [Ca²⁺]_i was 1.0-1.6 µM and there was a dense central cataract. Conclusions: Our results suggest there is a circulation of Ca²⁺ in the lens, with passive leak into the lens occurring across fiber cell membranes and active extrusion across surface cell membranes. The movement of Ca²⁺ from the central to surface cells is via gap junctional coupling, which is therefore essential for Ca²⁺ homeostasis in the central mature fibers.

Keywords: calcium • gap junctions/coupling • gene/expression