Functional Study of the Possible Phosphorylation Sites in the Human CLCN1 Cl- Channel via Heterologous Expression System.

• Main Authors: Ren-Yu, 黃仁裕
• Other Authors: Min-Jon Lin
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/34984986973434160915

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 96 === CLCN1 voltage-dependent chloride channel play an important role in membrane potential repolarization of mammalian skeletal muscle. Mutations in human CLCN1 lead to the myotonia congenital which is an inherited human disease. It has been known protein kinase C (PKC) can modulate the CLCN1 channel, however, the phosphorylated sites are still unclear. The purpose of this study was to investigate the possible phosphorylation sites in the human CLCN1 Cl- channel. The human CLCN1 cRNA was in vitro transcribed from linearized hCLCN1/pTLN plasmid, and then inject it into the Xenopus laevis oocytes using a microinjector. Two-electrode voltage clamp (TEVC) technique was used for the measurement of ionic current of human CLCN1. In the present study we construct 21 mutant recombinant plasmids by use the technique of site-directed mutagenesis. All mutants were designed according to the putative phosphorylation sites in C-terminus of CLCN1. Human CLCN1 and mutants were transient expressed in Xenopus oocytes by the microinjection of CLCN1 cRNA. To test whether mutant proteins lacking sensitivity to protein kinase C, an activator of PKC phorbol 12-myristate 13-acetate (PMA, 2 μM) was used in this study. The functional effects of PMA on wild-type and various mutants were determined by using TEVC recording from Xenopus oocytes. The treatment with PMA causes an inhibiting effect of wild-type CLCN1 current and a shift of the half-maximum activation of open probability (V1/2) to more positive membrane potentials (from -42.9 12.5 to -13.7 5, n = 8). These shifted-effects of most CLCN1 mutants by treated with PMA are similar to the wild-type, including S682A (from -32.9 4.2 to -15.9 10.2, n = 4), S720A (from -26.8 5.7 to -5.2 12.1, n = 4), S740A (from-55.7 13.5 to -24.2 9.1, n = 4), T782A (-40.4 10.3 to -8.1 11.5, n = 4), T891A (from -36.5 9.2 to -14.8 6, n = 6), S892A (from -27.5 6.8 to -1.3 10.4, n = 4) and S896R (from -45.4 13.4 to -13.7 12.9, n = 8). As so far only the S892P (from -39.5 11.2 to -35.7 14, n = 6) and a double mutant S892A/T893A (from -45.2 4.4 to -38.4 4.4, n = 4) were not change V1/2 by treated with PMA. In order to mimic phosphorylation at position 892, we made a mutant where a serine residue at 892 was substituted by aspartic acid. The result showed that the V1/2 of S892D was more positive than the wild-type, but similar to wild-type in the presence of PMA (-10.2 9.7 vs -13.7 5.0). Furthermore, the treatment of S892D with PMA did not change the value of V1/2 (from -10.2 9.7 to -9.6 6.9, n = 4). These results suggest that sequence including serine 892 has functional phosphorylated site for protein kinase, and therefore to modulate the functional properties of human CLCN1 channels.