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Fuctional Expression of a Novel Mutation R675Q Identified in a Chinese Normakalemic Periodic Paralysis Family

Author: WuLei
Tutor: WuWeiPing
School: PLA Postgraduate Medical School
Course: Neurology
Keywords: human muscle sodium channel a subunit(hSkM1) R675Q human embryonic kidney epithelial cell(HEK293) site-directed mutagenesis RT-PCR Western-blot clamp-patch activation slow inactivation
CLC: R746
Type: PhD thesis
Year: 2007
Downloads: 60
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Abstract


Objective construct R675Q human skeletal muscular sodium channel(pRcCMV -R675Q-SCN4A)in vitro,tranfect human embryonic kidney epithelial cell(HEK-293)by calcium phosphate precipitation method,investigate the characteristic of Na current’s Electrophysiology, further explain period paralysis’s pathophysiology ,make base for its moleculer diagnosis and more effective therapy. Methods designed primers that include mutational base,cDNA encoding the adult isoform of the human muscle sodium channel a subunit (hSkM(?)) was used as a template for in vitro site-directed mutagenesis,constructed pRcCMV-R675Q -SCN4A. the plasmid was extracted by Wizard Plus Maxipreps DNA Purification System and transiently transfected into HEK293 cell lines with Lipofectamin2000 .24h and 48h after transfection, expression levels of SCN4A were detected by RT -PCR and Western-blot. Whole cell voltage-clamp recording was used to study gating characteristics of the channels. Results:1. the site-mutagenesis in pRcCMV-R675Q-SCN4A was confirmed by sequencing.2. 24h and 48h after transfection,expression of SCN4A was detected by RT-PCR, the relative light density of non-transfection is 0.34±0.044,24h after transfection,it’s 0.72±0.05,48h after transfection,it’s 0.79±0.04;it’s statistically significant between transfection and non-transfection( p <0.01). 24h and 48h after transfection,expression of SCN4A was detected by Western-blot, the relative light density of non-transfection is 0.09±0.01,24h after transfection,it’s 0.57±0.06,48h after transfection,it’s 1.15±0.04;it’s statistically significant between transfection and non-transfection( p <0.01).3. On average, cells transfected with wild-type(WT) hSkM(?) cDNA had largerer peak Na currents (1798.9±106.5pA; n=35) than did those transfected with R675Q (1035.6±57.3pA; n=22), the difference was statistically significant (p<0.01).the fast inactivation properties of the R675Q current appeared to bequalitatively identical to that of wild type, The small variation in ζ observed for R669H was not statistically different from that of WT.for the fast activation,The peak conductance-voltage relationship of R675Q channels had a leftward shift, estimated to be a 7 mV depolarized shift in the midpoint of the fitted curve in comparison with that of wild type.and this shift was statistically significant (p <0.01).4. The voltage dependence of slow inactivation was determined by using a 40 sec conditioning pulse, which was sufficiently long to allow slow inactivation to approach steady state;Slow inactivation was enhanced by R675Q. The steady-state voltage dependence for R675Q was shifted by 29 mV in the hyperpolarized direction compared with WT channels.5. The rate of entry to the slow-inactivated state was measured by the use of a conditioning pulse of varying duration. A 20 msec return to Vhold was interposed between the conditioning and test pulses. The extent of slow inactivation was greater for R675Q as shown by the lower relative INa after long conditioning pulses.the time constant of R675Q is 7.93s±0.58s,the WT is 2.83±0.15 s,the time course is statistically significant (p <0.01)between them.6. We designed protocols to examine the recovery of channel availability after a wide range of conditioning pulse Durations.the conditioning pulse of durations between 40 msec and 60 sec. The time constant for recovery from fast inactivation at 40ms conditioning pulse duration was similar for WT (5.77±1.62ms; n = 6) and R675Q(3.47±0.87ms; n =6) channels,there is not statistically significant between them(p>0.05).After a 1 sec conditioning pulse,the fast inactivated fraction for WT (2.48±0.22ms; n = 6) and R675Q(2.99±0.76ms; n =6) channels, there is not statistically significant between them(p>0.05);The second component is median fraction,it recovered more slowly for R675Q (357.08±17.08ms; n= 6 )than for WT(67.11±5.01ms; n =6) channels. After 60 sec conditioning pulse ,The median fraction of time course like the data after a 1 sec conditioning pulse, recovery of this component was impeded for R675Q (658.9±78.09ms; n= 6) compared with WT (180.2±12.12ms; n= 6); The second component is slow fraction, Recovery of this component is also prolonged by R675Q (16.3±3.08s; n= 6) compared with WT (6.96±0.8s; n=6).7. A sequential protocol with a single conditioning pulse followed by a series of short test depolarizations was used to monitor the time course of recovery after 240 sec conditioning pulse.sdata from this protocol show a slowing for the recovery of the median component of R675Q (654.9±93.5ms; n = 6) compared with that of WT (273.9±48.2ms; n=6);The slow fraction was also slowed for R675Q (47.8±4.9s ; n=6)compared with that of WT(21.3±2.4s; n=6).Conclusion:1. successfully constructed the cell model of period paralysis that carry R675Q mutation.2. confirmed the expression of SCN4A’s gene and protein after transfection by RT-PCR and Western-blot.3. The mutation shifted activation in the depolarizing direction but had little effect on fast inactivation and its recovery,it dramatically impaired slow inactivation,prolonged slow inactivation and impeded recovery from slow inactivation;suggest that the S4 segment in domain II maybe involved in fast activation and slow inactivation Converge.

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