Association of Whirlin with Ca_v1.3 (α_1D) Channels in Photoreceptors, Defining a Novel Member of the Usher Protein Network

Abstract

PURPOSE.Usher syndrome is the most common form of hereditary deaf-blindness. It is both clinically and genetically heterogeneous. The USH2D protein whirlin interacts via its PDZ domains with other Usher-associated proteins containing a C-terminal type I PDZ-binding motif. These proteins co-localize with whirlin at the region of the connecting cilium and at the synapse of photoreceptor cells. This study was undertaken to identify novel, Usher syndrome-associated, interacting partners of whirlin and thereby obtain more insights into the function of whirlin. METHODS.The database of ciliary proteins was searched for proteins that are present in both the retina and inner ear and contain a PDZ-binding motif. Interactions with whirlin were evaluated by yeast two-hybrid analyses and validated by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization in the retina with immunofluorescence and immunoelectron microscopy. RESULTS.The L-type calcium channel subunit Ca v 1.3 (␣ 1D ) specifically interacts with whirlin. In adult photoreceptors, Ca v 1.3 (␣ 1D ) and whirlin co-localize in the region of the connecting cilium and at the synapse. During murine embryonic development, the expression patterns of the Whrn and Cacna1d genes show significant overlap and include expression in the eye, the inner ear, and the central nervous system. CONCLUSIONS. The findings indicate that Ca v 1.3 (␣ 1D ) is connected to the Usher protein network. This conclusion leads to the hypothesis that, in the retina, whirlin scaffolds Ca v 1.3 (␣ 1D ) and therefore contributes to the organization of calcium channels in the photoreceptor cells, where both proteins may be involved in membrane fusions. (Invest Ophthalmol Vis Sci.

Cav1.4 IT mouse as model for vision impairment in human congenital stationary night blindness type 2

“…The expression profiling supported findings that Cav1.4 but also Cav1.3 5 , 54 - 63 and Cav1.2 4 , 5 LTCCs are expressed in the retina. Interestingly, Cav1.3 channels were upregulated in IT.…”

Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities

“…Other recent findings describe interactions of myomegalin with the SANS CEN domain [131], SPAG5 (sperm-associated antigen 5, also called astrin) with the usherin cytoplasmic region [132], and Magi2 (membrane-associated guanylate kinase inverted-2) with the SANS SAM region [133]. However, caution is needed when data from screens of USH protein-interacting partners are interpreted physiologically, because some of the interactions have not been confirmed in vivo [134, 135]. …”

Current understanding of usher syndrome type II

“…Additionally, USH2A was found to associate with collagen IV and fibronectin (98, 99). For whirlin, it interacts in vitro with calmodulin-dependent serine kinase (CASK) (100), a voltage-dependant calcium channel α subunit (Cav1.3) (101), and a transmembrane protein (NGL-1) (77). Therefore, all these proteins are candidate components in the USH2 complex at the PMC in photoreceptors and the ankle-link in hair cells (Figure 4).…”