D810E mutation depleted the kinase activity of c-KIT protein and induced the destruction of the inner ear structure
The DFG motif flipped between the “on” and “off” states in the activation process of the c-KIT protein(Mol et al., 2004; Mol et al., 2003). Asp810 was directed towards the positively charged guanidinium group of Arg815 in the autoinhibited conformation and coordinated the Mg2+ ion bridging of the α- and β-phosphates in the active conformation (Fig. 4a). Therefore, we deduced that the substitution of Asp810 with Glu810 elongated the side chain at this specific site and exerted a deleterious impact on the kinase activity of the protein. This prediction was further confirmed by SIFT (https://sift.bii.a-star.edu.sg/) and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) (Supplemental Data). In addition, c-KIT has been reported to indirectly regulate the transcription activity of microphthalmia-associated transcription factor (MITF) through the MAP kinase machinery to affect the survival, proliferation and apoptosis of melanocyte derived intermediate cells(Hemesath, Price, Takemoto, Badalian, & Fisher, 1998; Ronnstrand, 2004). RT-PCR revealed the downregulation of MITF-regulated genes (PAX3 , TYR , DCT , and MC1R ) (Fig. 4b), implying the reduction and dysfunction of the mutant KIT protein activity. However, the D810E mutation did not affect the transcription of KIT , MITF , or SOX10 .
To investigate the pathology of hearing loss, we dissected the cochleae from wild-type and mutant pigs for microcomputed tomography (micro-CT) scanning and three-dimensional reconstructions to observe the cochlear structure and integrity. The three-dimensional structure of the cochlea showed three semicircular canals perpendicular to each other and three and one-half turns of the cochlear capsule; any marked difference was not detected between wild-type and mutant pigs (Fig. 4c). The results of celloidin embedding and hematoxylin-eosin staining (CE-HE) showed that, in wild-type pigs, the organ of Corti, the Reissner’s membrane (RM), spiral ganglion (SG), tectorial membrane (TM), and the stria vascularis (SV) were arrayed normally in the cochlear duct (Fig. 4d). However, an extensive collapse of Reissner’s membrane onto the SV and the disordered organ of Corti were observed inc-KITc.2430T>A/+ pigs, which induced the profound hearing loss.