Herg Missense Mutation A422t And H562p Lead To Cell And Molecular Mechanism Of Lqt Syndrome

Human-ether-a-go-go-related gene (HERG) encodes the protein of delayed rectifier potassium channel that mediates IKr which contributes to the repolarization of the cardiac action potential. HERG protein is a pharmacological target for the majority of acquired LQTS as a result of drug blockade. While loss of functuion caused by HERG mutations are linked to hereditary LQTS (LQT2). So far, there are more than 200 LQT2 caused mutations were identified, but the mechanisms underlying the dysfunction of HERG channel are different.Two HERG missense mutants A422T and H562P, have been identified from two Chinese families with LQTS, resulting in loss of function. The present study was designed to investigate the molecular mechanisms underlying the loss of function caused by these two mutations using electrophysiological recording combined with molecular and cell biological assays. Results are as follows:1. Whole-cell currents recorded from the cells expressing A422T or H562P were dramaticlly reduced compared with WT, particularly in its tail, indicating loss of function. No matured protein of either A422T or H562P mutant was detected by western blot analysis, suggesting trafficking defects in mutate protein. In consistant with this，the co-localization of the A422T or H562P mutant with ER was further evidenced by immunocytochemistry staining, illustrating their ER retention.2. Both A422T and H562P exerted dominant negative suppression on WT in a concentration-dependent manner, resulting in the alteration in the kinetics processes, including activation, inactivation, deactivation, as well as recovery from inactivation as evidenced by patch-clamp recording. Paraleled with current reduction, the matured protein expression was much less than that of WT as shown in western blot analysis. The reduction of protein expression and alteration of channel kinetics of gating may account for the functionanl defects in HERG.3. Both the reduction of current and defect of protein trafficking in A422T mutant were partially rescued by incubating cells at lower temperature of 27℃, or with l0μM E4031, a HERG channel blocker, for 24 hr, indicating that A422T mutant was a mild misfolding protein and capable of assembling into tetramers as native state. But this was not the case for H562P mutant.Hsp70 or/and Hsp90 were co-expressed with A422T or H562P mutant in HEK 293 cells. An interaction between either Hsp70 or Hsp90 and channel protein was do identified by co-immunoprecipitation detection. The results also showed that the amount of Hsp70 or Hsp90 interacted with HERG protein was different between the WTs and the mutations". The protein expression and the amplitudes of current detected from WT were all enhanced in response to either co-expression of Hsp70 or that of Hsp90. Moreover, the reduction of currents in A422T mutant were partly rescued by co-expression of Hsp90 alone, but not Hsp70, although the protein expression of A422T was not improved significantly. The rescue effect of Hsp90 was abloished by celastrol, an inhibitor of Hsp90. When co-expressed Hsp70, Hsp90 and A422T, the amplitudes of current were not futher increased by adding Hsp70. Again, no rescue effects on both H562P current and protein expression were observed by overexpression of chaperons.Then, hypoxia stimulation (2%O2) was used to test the effect of ER stress on mutant protein folding. The matured protein expression and currents of WT were increased under the condition of 2% O2 incubation for 24 hr. Whereas only the function of A422T was partly rescued while the protein expression was not significantly affected. This indicated that the mis-folding protein could be rescued by the moderate ER stress. Same as other treatments, no any rescue effects were documented for H562P mutant.4. In order to study whether A422T or H562P protein retained in ER could lead to ER stress, the splicing mRNA of XBP1 was detected by RT-PCR. The results showed that there was no ER stress caused by the retaining of A422T or H562P protein. Rather, the retained proteins in ER were degradated in time by the ubiquitin-protease pathway, as shown in western blot analysis. Conclusions:1. The molecular mechanisms for LQT2 caused by A422T or H562P mutation were attributed to trafficking defects of the channel proteins in ER.2. The trafficking defect in A422T could be rescued by all tested methods in this study, reflecting that A422T mutant underwent a mild mis-folding and it was capable of assembling into native state tetramers in response to different treatments.3. In contrast to A422T, there was no any effective treatment to rescue the trafficking defect in H562P mutant. This might attribute to the location of H562P in the S5 segment which lined in the pore region of HERG potassium channels, which played key role in the conformation of protein. The interaction between H562P mutate protein and the molecular or pharmacological chaperons could be influenced by the change of protein conformation.