Association of whirlin with Cav1.3 (alpha1D) channels in photoreceptors, defining a novel member of the usher protein network.
OBJECTIVE
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 (alpha(1D)) specifically interacts with whirlin. In adult photoreceptors, Ca(v)1.3 (alpha(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 (alpha(1D)) is connected to the Usher protein network. This conclusion leads to the hypothesis that, in the retina, whirlin scaffolds Ca(v)1.3 (alpha(1D)) and therefore contributes to the organization of calcium channels in the photoreceptor cells, where both proteins may be involved in membrane fusions.
Kersten
Ferry F J
FF
Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
van Wijk
Erwin
E
van Reeuwijk
Jeroen
J
van der Zwaag
Bert
B
Märker
Tina
T
Peters
Theo A
TA
Katsanis
Nicholas
N
Wolfrum
Uwe
U
Keunen
Jan E E
JE
Roepman
Ronald
R
Kremer
Hannie
H
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019295:Computational Biology; D030562:Databases, Protein; D017403:In Situ Hybridization; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D055212:Photoreceptor Connecting Cilium / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012160:Retina / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C485995:Cacna1d protein, mouse; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D012333:RNA, Messenger; C477219:WHRN protein, human