Characterization of a Mutation in the Lens-specific MP70 Encoding Gene of the Mouse Leading to a Dominant Cataract

Mutations within connexin50 (Cx50) have been linked to various cataract phenotypes. To determine the mechanism behind cataract formation we used the paired Xenopus oocyte system in conjunction with transfected HeLa cells and genetically engineered mouse models to examine the functional characteristics of gap junctions in which a cataract-causing mutant of Cx50 (hereafter referred to as Cx50-S50P) is expressed. Channels comprising Cx50-S50P subunits alone failed to induce electrical coupling. However, the mixed expression of Cx50-S50P and wild-type subunits of either Cx50 or Cx46 – to create heteromeric gap junctions – resulted in functional intercellular channels with altered voltage-gating properties compared with homotypic wild-type channels. Additionally, immunofluorescence microscopy showed that channels of Cx50-S50P subunits alone failed to localize to the plasma membrane – unlike channels composed of Cx46 subunits, which concentrated at cell-cell appositions. Cx50-S50P colocalized with wild-type Cx46 in both transfected HeLa cells in vitro and mouse lens sections in vivo. Taken together, these data define the electrophysiological properties and intracellular targeting of gap junctions formed by the heteromeric combination of Cx50 or Cx46 and Cx50-S50P mutant proteins. Additionally, mixed channels displayed significantly altered gating properties, a phenomenon that may contribute to the cataract that is associated with this mutation.

Section: Journal Of Cell Science 120 (23)mentioning

confidence: 99%

Section: Journal Of Cell Science 120 (23)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

The cataract-inducing S50P mutation in Cx50 dominantly alters the channel gating of wild-type lens connexins

DeRosa

Xia

Gong

et al. 2007

“…The central role played by gap-junction-mediated cell-cell communication in lens physiology was underscored by the discovery that mutations in fiber cell connexins can lead to cataracts. Mutations in α8 result in dominant cataracts in mice (Chang et al, 2002;Graw et al, 2001;Steele et al, 1998) and humans Pal et al, 2000), and α3 mutations cause autosomal dominant congenital human cataract Rees et al, 2000). Similarly, targeted deletion of α3 or α8 in mice leads to electrical uncoupling of fiber cells and opacification of the tissue (Baldo et al, 2001;Gong et al, 1997;Gong et al, 1998;White et al, 1998).…”

mentioning

confidence: 99%

Development of a macromolecular diffusion pathway in the lens

Shestopalov

Bassnett

2003

The mammalian lens consists of an aged core of quiescent cells enveloped by a layer of synthetically active cells. Abundant gap junctions within and between these cell populations ensure that the lens functions as an electrical syncytium and facilitates the exchange of small molecules between surface and core cells. In the present study, we utilized an in vivo mouse model to characterize the properties of an additional pathway, permeable to macromolecules, which co-exists with gap-junction-mediated communication in the lens core. The TgN(GFPU)5Nagy strain of mice carries a green fluorescent protein (GFP) transgene. In the lenses of hemizyous animals, GFP was expressed in a variegated fashion, allowing diffusion of GFP to be visualized directly. Early in development, GFP expression in scattered fiber cells resulted in a checkerboard fluorescence pattern in the lens. However, at E15 and later, the centrally located fiber cells became uniformly fluorescent. In the adult lens, a superficial layer of cells, approximately 100 μm thick, retained the original mosaic fluorescence pattern, but the remainder, and majority, of the tissue was uniformly fluorescent. We reasoned that at the border between the two distinct labeling patterns, a macromolecule-permeable intercellular pathway was established. To test this hypothesis, we microinjected 10 kDa fluorescent dextran into individual fiber cells and followed its diffusion by time-lapse microscopy. Injections at depths of >100 μm resulted in intercellular diffusion of dextran from injected cells. By contrast, when injections were made into superficial fiber cells, the injected cell invariably retained the dextran. Together, these data suggest that, in addition to being coupled by gap junctions, cells in the lens core are interconnected by a macromolecule-permeable pathway. At all ages examined, a significant proportion of the nucleated fiber cell population of the lens was located within this region of the lens.

“…To date, ␣8 mutations have been reported in the N-terminal domain (G22R and R23T) (Chang et al, 2002;Willoughby et al, 2003), the E1 loop (D47A, E48K, V64G and V64A) (Steele et al, 1998;Berry et al, 1999;Zheng et al, 2005;Graw et al, 2001), the second transmembrane domain (P88S) (Shiels et al, 1998), and the C-terminal intracellular domain (I247M) (Polyakov et al, 2001). Mutations of ␣3 have been identified in the first transmembrane domain (F32L) (Jiang et al, 2003), E1 loop (P59L and N63S) (Bennett et al, 2004;Mackay et al, 1999), E2 loop (P187L and N188T) (Rees et al, 2000;Li et al, 2004) and C-terminal domain (S380fs) .…”

Section: Figmentioning

confidence: 99%

Knock-in of α3 connexin prevents severe cataracts caused by an α8 point mutation

Xia

Cheung

DeRosa

et al. 2006

. Knock-in of ␣3 connexin prevents severe cataracts caused by an ␣8 point mutation. J. Cell Sci. 119, 2138-2144.We apologise for an error that occurred in the heading of Table 2, which wrongly stated 'heteromeric' instead of 'homotypic' and 'heterotypic' instead of 'heteromeric'. This error appeared in both the print and the online versions of this article. The correct version is shown below. G jmin represents the minimum conductance value, V 0 indicates the transjunctional voltage value midway through the G j decline, and A denotes the cooperativity constant, reflecting the number of charges moving through the transjunctional field. Signs + and -for V j indicate transjunctional potential polarity.