The Regulatory Function Of The Four And A Half LIM Domains Protein2on Mutant Herg Channel And Its Machanism

BACKGROUND:The human ether-a-go-go related gene (HERG) encodes the pore-forming subunit of the rapid component of the delayed rectifier K channel, Kv11.1, which are expressed in the heart, various brain regions, smooth muscle cells, endocrine cells, and a wide range of tumor cell lines. However, it is the role that Kv11.1channels play in the heart that has been best characterized, for two main reasons. First, it is the gene product involved in chromosome7-associated long QT syndrome (LQTS), an inherited disorder associated with a markedly increased risk of ventricular arrhythmias and sudden cardiac death. Second, blockade of Kv11.1, by a wide range of prescription medications, causes drug-induced QT prolongation with an increase in risk of sudden cardiac arrest. These channels are variously referred to as IKr, HERG, or Kv11.1. HERG when referring to the channel protein, Kv11.1when referring to the fully assembled channels studied in heterologous expression systems, and IKr when discussing the native channel. LQTs can be classified twelve subtypes of LQT1-LQT12, which LQT2is the most common clinical subtype in China. Loss-of-function mutations in membrane-spanning proteins usually result in protein misfolding, which leads to enhanced degradation and hence decreased levels of functional protein at the plasma membrane. HERG K-channels are tetrameric complexes with each subunit containing cytoplasmic N-terminal and C-terminal domains and a transmembrane region that forms the ion-conducting pore as well as containing the voltage-sensor domains. The cytoplasmic regions contain structurally distinct domains, including an N terminal PAS (Per/Arnt/Sim) domain and a C-terminal cyclic nucleotide homology binding domain that help to regulate function and contribute to channel assembly. There are two main isoforms of HERG, referred to as HERGla and HERGlb. The HERGlb isoform lacks the N-terminal PAS domain as well as most of the remaining N-terminal cytoplasmic region. It is thought that the functional channels in the human heart are heterotetramers composed of two HERGla and two HERGlb subunits, although it is possible that it may be a mixture of HERG1a/HERG1b heterotetramers and HERG1a homotetramers. It is estimated that at least80%of missense mutations in KCNH2result in defects in assembly and hence trafficking of functional channels to the plasma membrane. Despite the similarity in the underlying molecular mechanism there is, however, a wide range of clinical phenotypes associated with the different mutations. Although considerable work has been undertaken looking at pathways of HERG trafficking and the role of chaperones in detecting misfolded mutant proteins, the question of why the outcomes are so variable for different mutants has not been addressed. Cells contain robust systems for facilitating the folding and assembly of proteins and for recognizing misfolded proteins, which are then targeted for degradation. The commonest generic mechanism for recognition of misfolded proteins is the exposure of hydrophobic regions, which may be due to unfolding of domains, thereby exposing residues normally buried in the core and/or from disruption of domain-domain interfaces (either intra subunit or inter subunit) that exposes hydrophobic surfaces that would normally be buried by the interface. The HERG PAS domain is the only domain of the channel that we have atomic level structural information for. This domain is also involved in domain-domain interactions within the channel and is a hotspot for mutations that cause congenital LQTS2. It is, therefore, a logical place to start any investigation into the structural basis of how different mutations affect assembly and trafficking of the channels.In a previous study, by Yeast two-hybrid technique, we found15proteins interacting with HERG potassium channel, which contains protein tyrosine phosphatase non-receptor type12(PTPN12),caveolin-1, FHL2with the LIM domain and so on, meanwhile by using co-immunoprecipitation, immunofluorescence and other technique, we confirmed FHL2has an important role in the regulation of HERG potassium channel.FHL2protein may be as scaffolding protein, making HERG combine some signal transduction protein, receptor protein, protein structure and so on.Loss-of-function mutations in membrane-spanning proteins usually result in protein misfolding, which leads to enhanced degradation and hence decreased levels of functional protein at the plasma membrane.In this study, we want to find out that FHL2can regulate mutant cardiac HERG potassium channels or not, especially HERG N-terminal PAS area, in order to further understand the mechanism of HERG potassium channels and provide new theoretical basis in the process of clinical treatment to develop new drugs for the treatment of LQTs.OBJECTIVE:To study the regulatory function of the four and a half LIM domains protein2on mutant HERG channel and its mechanism.METHODS:(1) Use two-step PCR site-directed mutation technology in vitro to constuct pcDNA3.0-HERG NT373plasmid, pcDNA3.0HERG-CT159plasmid, pcDNA3.0-HERG-Y43C plasmid, pcDNA3.0HERG-F29L plasmid, pcDNA3.0HERG-R56Q plasmid, pcDNA3.0HERG-I96T plasmid and their eGFP plasmid, as well as pcDNA3.1-FHL2-RFP plasmid and pEGFP-FHL2plasmid;(2) With Lipofatamine2000, the mutant HERG plasmid transfection and HERG plasmid alone with FHL2plasmid transfection into HEK293cells, successfully establish HEK293cells heterologous expression system transfected by mutant HERG plasmid alone and co-transfected by mutant HERG plasmid and FHL2plasmid;(3) Use patch clamp technique, under the whole-cell recording, to record mutant heart HERG potassium channel current. Observe the HERG potassium channels currents in the following six groups:the first group is HEK293cells transfected pcDNA3.0HERG-NT373and it with FHL2; The second group is HEK293cells transfected pcDNA3.0HERG-CT159and it with FHL2; The third group is HEK293cells transfected pcDNA3.0HERG-Y43C and it with FHL2; The fourth group is HEK293cells transfected pcDNA3.0HERG-F29L and it with FHL2; The fifth group is HEK293cells transfected pcDNA3.0HERG-R56Q and it with FHL2; The sixth group is HEK293cells transfected pcDNA3.0HERG-I96T and it with FHL2.RESULTS:(1) successfully constuct pcDNA3.0-HERG NT373plasmid, pcDNA3.0HERG-CT159plasmid, pcDNA3.0-HERG-Y43C plasmid, pcDNA3.0HERG-F29L plasmid, pcDNA3.0HERG-R56Q plasmid, pcDNA3.0HERG-I96T plasmid and their eGFP plasmid, as well as pcDNA3.1-FHL2-RFP plasmid and pEGFP-FHL2plasmid, verified by gene sequencing;(2) Successfully establish HEK293cells heterologous expression system transfected by mutant HERG plasmid alone and co-transfected by mutant HERG plasmid and FHL2plasmid;(3) With patch clamp technique, by the mode of whole cell recording, six mutant HERG single transfection and their with FHL2co-transfection:①pcDNA3.0HERG-ΔNT373AA transfection group and pcDNA3.0HERG-Δ NT373AA-FHL2, co-transfection group, pulse current maximum (n=12, P=0.51), pulse current density (n=12, P=0.15), tail current maximum (n=12, P=0.08), tail current density (n=12, P=0.21), pulse current voltage (n=12, P=0.44), time constant of deactivation (n=12, P=0.13) were not statistically significant that can be considered that two groups were the same;②pcDNA3.0-HERG-ΔCT159AA single transfection group and pcDNA3.0-HERG-ΔCT159AA-FHL2co-transfection group, pcDNA3.0HERG-ΔCT159AA-FHL2co-transfection group were increased statistically compared with pcDNA3.0-HERG-ΔCT159AA single transfection group in maximum pulse current (n=11, P<0.05), pulse current density (n=11, P<0.05), maximum of tail current (n=11, P<0.05), tail current density (n=11, P<0.05), time constant of deactivation (n=11, P<0.05), and voltage of maximum of pulse current decreased slightly(n=11, P0.67);③pcDNA3.0-HERG-Y43C single transfection group and pcDNA3.0HERG-Y43C-FHL2co-transfection group, two groups are unable to detect the current;④pc-DNA3.0-HERG-F29L single transfection group and pcDNA3.0-HERG-F29L-FHL2co-transfection group, pcDNA3.0HERG F29L-FHL2co-transfection group were increased statistically compared with pcDNA3.0-HERG-F29L single transfection group in maximum of pulse current (n=10, P<0.05), pulse current density (n=10, P<0.05), maximum of tail current (n=10, P<0.05), tail current density (n=10, P<0.05), while voltage of maximum of pulse current (n=10, P<0.05) and the time constant of deactivation (n=10, P<0.05) significantly decreased;⑤pc-DNA3.0-HERG-R56Q single transfection group and pcDNA3.0-HERG-R56Q-FHL2co-transfection group, pcDNA3.0HERG R56Q-FHL2co-transfection group were increased statistically compared with pcDNA3.0-HERG-R56Q single transfection group in maximum of pulse current (n=10, P<0.05), pulse current density (n=10, P<0.05), maximum of tail current (n=10, P<0.05), tail current density (n=10, P<0.05), while voltage of maximum of pulse current (n=10, P<0.05) and the time constant of deactivation (n=10, P<0.05) significantly decreased;⑥pc-DNA3.0-HERG-I96T single transfection group and pcDNA3.0-HERG-I96T-FHL2co-transfection group, pcDNA3.0HERG I96T-FHL2co-transfection group were increased statistically compared with pcDNA3.0-HERG-I96T single transfection group in maximum of pulse current (n=10, P<0.05), pulse current density (n=10, P<0.05), maximum of tail current (n=10, P<0.05), tail current density (n=10, P<0.05), while voltage of maximum of pulse current (n=10, P=0.067) and the time constant of deactivation (n=10, P=0.89) decreased without statistical significance.CONCLUSIONS:FHL2protein may regulate the channel function through the N terminal of HERG; In heterologous expression system, FHL2protein can significantly increase current amplitude of HERG-F29L, HERG-R56Q, HERG-I96T potassium channels; but for HERG-Y43C, FHL2protein almost has no effect. This provides new thought for the severity of HERG gene mutatins with clinical classification and treatment of FHL2on LQTS effect.