Functional Analysis Of Pendrin (SLC26A4) And Its Pathogenic Mutations In Cos-7Cells

Diseases caused by SLC26A4mutations are inherited in an autosomal recessive manner. More than200SLC26A4mutations have been identified that are associated with both Pendred syndrome and with DFNB4(OMIM600791), which causes non-syndromic hereditary deafness. People with Pendred syndrome are deaf and often have an enlarged thyroid gland (thyroid function is normal or slightly lower). Deafness caused by SLC26A4mutations are often accompanied by inner ear malformations, the most common of which are an enlarged vestibular aqueduct (EVA) and Mondini deformity. Pendrin in the inner ear takes part in Cl" and HCO3-ion transport to maintain cochlear homeostasis. Additionally, pendrin do not mediate the transport of potassium ion across cell membranes directly.However, The study found Pendrin have played a crucial role in auditory.by maintainning the endocochlear potential (equilibrium potential potassium). The deafness-causing mutation S448X in pendrin was first discovered in our laboratory. In this study, we transfected WT-EGFP and S448X-EGFP plasmids into cos-7cells to detect their expression. We immunostained the endoplasmic reticulum (ER), Golgi apparatus and microtubule network in order to determine the distribution of the pendrin protein in these organelles. Furthermore, to explore the effect of S448X on chloride ion transport ability in cells, we recorded the current differences between cells expressing WT and S448X proteins with patch clamp technology.Objective:To investigate the mechanism of the deafness-causing mutation S448X in the pendrin protein through measurement of the expression levels and functional activities of the wild-type (WT) and mutant proteins. Methods:pEGFP N1SLC26A4S448X (S448X-EGFP) and wild-type pEGFP N1SLC26A4(WT-EGFP) were transfected into cos-7cells and their expression detected by western blotting. Immunostaining and confocal microscopy were used to determine the subcellular localization of WT and S448X proteins. The effects of overexpression of WT and S448X pendrin on ion transport in the cell were measured by whole-cell patch clamp analysis.Results:1、The WT-EGFP and S448X-EGFP fusion proteins were expressed in cos-7cells, and the resulting bands on the SDS-PAGE gel were of the expected molecular weights (Fig.1A). The lower molecular weight observed for the S448X mutant is due to the fact that the codon for a serine at position448has been replaced with a stop codon and the resulting protein is truncated.2、S448X protein was expressed in the cytoplasm and colocalized with the ER, but not with the Golgi apparatus or the microtubule network. Wild-type pendrin was found mainly in the cell membrane, and the small amount in the cytoplasm was likely due to the high expression level of the protein in the cell.3、G418screened COS-7cells stably expressing wild-type SLC26A4encoding Pendrin protein. Photograph of confocal laser scanning microscope, observed in the COS-7cytoplasm and cell membrane were observed in the expression of green fluorescent protein, GFP expression is clear. In the S448X mutant plasmid transfected COS-7cell, after10days screening cells all died,and no screening monoclonal cells in G418.4、Statistical analysis showed that there was a significant difference (P<0.05) in current amplitude between cells expressing WT and S448X at each clamping voltage, and control cells (untransfected cos-7cells) had a similar curve to the cells expressing the S448X mutant protein (P>0.05). The current amplitudes of cells expressing WT and S448X and the control cells were significantly reduced (P<0.05)20min after addition of NPPB, a chloride ion inhibitor. 5、The expression of SLC26A4in wild type and S448X mutant cells can be recorded to the potassium ion current stability. Statistical analysis showed that there was a significant difference (P<0.05) in current amplitude between cells expressing WT and S448X at clamping voltage:30,50,70,90mV, and control cells (untransfected cos-7cells) had a similar curve to the cells expressing the S448X mutant protein (P>0.05). The current amplitudes of cells expressing WT and S448X and the control cells were significantly reduced (P<0.05)20min after addition of TEACL,a potassium ion inhibitor. Through the analysis of Ⅰ-Ⅴ curve, when the clamping voltage groups in-90-10mV potassium current amplitudes were not significantly increased, there was no significant difference between each group statistics (P>0.05); when the voltage at the30-90mV, with the depolarization of the membrane potential, amplitude of the potassium ion current increases, the Ⅰ-Ⅴ curve closer to the Y axis, showing the obvious characteristics of the outward rectification.Conclusion:Immunofluorescence showed wild-type pendrin protein mainly expressed in the cell membrane, mutant S448X expressed mainly in the endoplasmic reticulum, patch clamp experiments showed that mutation had a reduced ability to transport ions. The study shows that SLC26A4gene mutation mechanism induced deafness, namely through the effect of pendrin protein transport, it can not reach the formation of cell membrane, so that the anion transport affected, while SLC26A4gene mutation can affect cell outwardly rectifying potassium ion channel activity. This may be an important cause of SLC26A4gene mutations lead to deafness and enlarged vestibular aqueduct.