Mechanisms Of KCNE Subunits Modulating KCNQ1 Channels And β2 Subunit Modulating Drosophila BK Channels

Ion channels are protein molecules that span across the cell membrane allowing the passage of ions from one side of the membrane to the other.Ion channels play critical roles in generating and facilitating the transmission of information,in the form of action potentials,within the CNS,PNS,and the cardiovascular system.The diverse functions of ion channels stem largely from different ancillary proteins.Any anomaly in these channels and ancillary proteins could lead to serious diseases.In this study,we focus on three types of ion channels:KCNQ1 potassium channel,large-conductance Ca²⁺-and voltage-activated potassium（BK）channel and T-type calcium channel.This paper is mainly divided into the following four parts around the three channels.（1）KCNE2β-subunit modulates plasma-membrane localization of KCNQ1 channel.KCNE2β-subunit has been reported to associate with KCNQ1 to reduce the surface expression and current amplitude of KCNQ1.However,the regulatory mechanisms remain elusive.To gain mechanistic insights into the interaction between KCNQ1 and KCNE2,we made a series of mutations in the N-terminus of KCNE2,and examined the influence of these mutants on the surface expression and functional properties of KCNQ1.Our results showed that a majority of KCNQ1/KCNE2 channel complexes appear to reside in the endoplasmic reticulum（ER）.In addition,we found that either mutation of the hydrophobic residues or deletion of the 25–29 segment（NWRQN）in the N-terminus of KCNE2 would partially weaken the inhibitory effect of KCNE2 on KCNQ1.Deletion of both the NWRQN sequence and hydrophobic residues nearly abolished the inhibitory effect of KCNE2.Therefore,we propose that KCNE2 alters the plasma-membrane localization of KCNQ1 via synergistic retention/retrieval signaling motifs made up of a novel structural motif（NWRQN）and hydrophobic residues within its N-terminal.Our study implicates a novel mechanism for KCNE2 to modulate the plasma-membrane localization of KCNQ1 and provides important clues for further exploring other voltage-gated potassium channels in the form ofα/βcomplexes.（2）Molecular mechanisms underlying KCNE subunits forward trafficking.Previous studies have revealed that trafficking defects of KCNE are related to the pathogenesis of LQTS.However,molecular mechanisms underlying KCNE forward trafficking remain elusive.In this study,we made a series of mutations of KCNE1 and KCNE2,and examined their subcellular localization and functional properties.We identified a highly conserved motif（[K/R]S[K/R][K/R]）in the proximal C-terminus,which regulates the endoplasmic reticulum（ER）export of KCNE1 and KCNE2 in HEK293 cells.Our results indicate that forward trafficking of KCNE2 controlled by the motif（KSKR）is essential for suppressing the surface expression and current amplitude of KCNQ1.Unlike KCNE2,the motif（RSKK）in KCNE1 plays important roles in modulating the gating of KCNQ1 in addition to mediating the ER export of KCNE1.We also demonstrated that the KCNE2C-terminus might not physically interact with KCNQ1 while KCNE1 C-terminus did.These results contribute to the understanding of the mechanisms of KCNE1 and KCNE2membrane targeting and how they coassemble with KCNQ1 to regulate channel activity.（3）Differentialβ2 subunit modulations between mammalian and Drosophila BK/β2channels.After associating with the auxiliaryβ2 subunit,mammalian BK/β2 channels（hSlo1/β2 or mSlo1/β2）exhibit enhanced activation and complete inactivation.However,how theβ2 subunit modulates the Drosophila BK channel remains elusive.In this study,by comparing the different functional effects on heterogeneous BK/β2 channels,we determined three different modulations between mammalian and Drosophila BK/β2channels:（1）The“inactivation ball”（FIW）inβ2 N-terminus is essential for the incomplete inactivation of dSlo1/β2,but may also function as an retention/retrieval signal of the channel complex.（2）The residues K33,R34,K35 inβ2 are responsible for the slow activation of dSlo1/Δ3-β2.（3）Compared to mSlo1/β2,the pre-inactivation of dSlo1/β2channel was enhanced.These results in our study provide insights into the different modulations ofβ2 subunit between mammalian and Drosophila BK/β2 channels and structural basis underlying the activation and pre-inactivation of other BK/βcomplexes.（4）Syntaxin-3A binds and regulates T-type calcium channels.Syntaxin-1A has been reported to bind and regulate T-Type calcium channels to control low-threshold exocytosis.Whether syntaxin-3A can bind and regulate T-type calcium channels remains unknown.Here,we report that syntaxin-3A can physically interact with T-type calcium channels and suppress the current density of these channels.This inhibitory effect is not due to the fact that syntaxin-3A could reduce the surface expression of T-type calcium channels.We found that syntaxin-3A can specifically bind the intracellular II-III linker region of Ca_v3.2channel.In addition,our results reveal that the transmembrane domain of syntaxin-3A is essential for the interaction with Ca_v3.2,but the key sites where it interacts with the channel need further study.Altogether,our findings demonstrate the interaction between syntaxin-3A and T-type calcium channels and provide insights into how syntaxin-3A binds and regulates calcium channels.