Molecular Analysis Of Inherited Cataract In A Family And The Gating Mechanism Of Gap Junction Formed By Cataract Related Gene-connexin50

Cataract remains a leading cause of blindness and visual impairment all over the world, accounting for 46% of all blindness, at least a third of all cases are familial. Since mutations in the 13 known genes (CRYAA, CRYAB, CRYBB1, CRYBB2, CRYBA1/A3, CRYGC, CRYGD, Connexin50, Connexin46, intrinsic membrane protein LIM2, cytoskeletal protein BFSP2, the major intrinsic protein-MIP and the heat shock factor-HSF4) have previously been demonstrated to be the frequent reason for isolated congenital cataracts. A unique cataract was observed in a 4-generation Chinese family, which characterized with autosomal dominant later-onset. We aimed to find the cataractgenesis gene mutations of these 13 genes in this family. We detected the mutations using polymerase chain reaction (PCR) and direct sequencing. Not any mutations were found in most of the 13 genes, except CRYBB2, BFSP2 and CRYGD. A transversional mutation in the intron of CRYBB2, some silent, mutations in the exons of BFSP2 and CRYGD have been found in the cataract family, but further study show that this can also be found in normal Chinese person. Though we did not search any cataract-associated mutations in the coding regions of these 13 genes, we can't exclude them from this family definitely, as some mutation may be located in non-coding regions of the genes. The results indicate that unidentified genes will underlie the occurrence of late onset cataract in this family. A genome-wide screening will be carried out in the next study.Gap junction channel provides a pathway for direct cell-to-cell communication of small molecules as large as 1.5 kDa and ions. It is important in maintain the stabilization of cellular environment and the regulation of various physiological process. Chemical gating of gap junction channels by intracellular pH may be an important mechanism for the physiological regulation of cell-cell coupling. In the lens, pH gating of gap junction channels has been implicated as a possible cause of cataract. Gap junction channels formed by connexin50 (Cx50) are critical for maintenance of eye lens transparency. Previous study indicated that the Cx50 channels showed sensitivity to the external Ca~2+ and low pH. The Cx50 channel was activated at lowexternal Ca2+ and blocked by intracellular acidification. Cleavage of the carboxyl terminus (CT) of Cx50 to produce truncated Cx50 (Cx50trunc) occurred naturally during maturation of lens fiber cells. The channels formed by Cx50trunc was found to be more sensitive that that formed by Cx50. Previous research had given detailed study on the electrophysiological character of Cx50, but the mechanism of its gating control is under confirmation. It has been suggested that calmodulin (CaM) participate in gating some kinds of gap junction. Here we performed confocal colocalization, co-immunoprecipitation, dye uptake-leakage assay and patch clamp experiments, to study the intra- and inter-molecular interactions among CT and CL (cytoplasmic loop) of Cx50 and CaM, and the role of these interactions in Cx50 hemichannel gating control.Firstly, we studied the intramolecular interactions and its role in mediating gap junctional localization of CaM. Results exhibited that the CaM could colocalize Ca2+-dependently with CT in the linear area of cell-to-cell contact formed by Cx50trunc, while it couldn't localize in the linear area without expression of CT. Further study indicated that the CT could interact Ca2+-independently with the cytoplasmic loop (CL) of Cx50. These data put forward the importance of Ca2+-independent intramolecular interaction between CT and CL of Cx50, which mediate the Ca2+-dependent binding of CaM to Cx50.In addition, we carried dye uptake-leakage assay and patch clamp experiments, to demonstrate the above hypothesis from functional properties Cx50 hemichannels. Results showed that, (1). Cx50-HeLa cells that had been loaded with LY were washed containing 2 mM Ca2+ extracellular solution, the high intensity of fluorescence was observed. The open probability of hemichannel decreased with the presence of 2 mM Ca2+, so LY retained in cytoplasm. (2) when LY preloaded Cx50-cells were washed and bathed with external solution with Ca2+-free, or the Cx50-HeLa cells were incubated with the bath solution containing 50 u g/ml W7 first, then loaded and washed LY with external solution containing 2 mM Ca2+, the fluorescence intensity was very low under these two conditions. It indicated that the CaM and Cai+ are critical factors in Cx50 hemichannel gating. (3). In the absence of Ca2+, the polarizing pulses applied to Cx50-HeLa induced characteristic slowly activating outward currents. However, when the Ca2+ in external solution was increased to 2 mM, the amplitude of the macroscopic hemichannel currents decreased to zero. It indicated that the open probability of hemichannel decreased because the presense of Ca +. When the Cx50-HeLa cells were incubated in extracelluar buffer with 2 mM Ca2+and 50 u. g/ml W7 (CaM inhibitor), the polarizing pulses induced characteristic slowly activating both outward and inward currents. It indicated the gating role of Ca2+ andCaM in voltage-dependent Cx50 hemichannels. The above results demonstrate that the interactions between CaM and Cx50 are critical in gating control of Cx50 gap junction channel.We also studied the possible mechanism of pH gating control in connexin50 hemichannel, using confocal colocalization, dye uptake-leakage assay and patch clamp technology. Results exhibited that, (1). Under pH6.5, Cx50-HeLa cells allowed dye to leak out when preloaded with LY and then washed in Ca2+-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca2+. It indcated that the Ca2+ was an important factor in pH gating of connexin50 hemichannel. The open probability of hemichannel decrease with the presence of 2 mM Ca2+, so LY retained in cytoplasm. (2). With no extracellular Ca2+, whole-cell recording revealed transmembrane outward currents sensitive to voltage. It indicated that the sensitivity to pH decreased in Cx50 hemichannels under this condition. (3). After being pretreated with W7, the Cx50-HeLa cells allowed dye to leak out when preloaded with LY and then washed in 2 mM Ca2+ external solution (pH 6.5). Further study exhibited that the CaM could localize in the gap junction formed by Cx50 under pH 6.5. The above results demonstrated the participation of CaM and Ca2+ in Cx50 channel gating, it may due to Ca2+-dependent binding of CaM to Cx50 gap junction.