KCNQ1 And KCNE1 In The I_Ks Channel Complex Make State-dependent Contacts In Their Extracellular Domains

The slow delayed rectifier (I_Ks) channel is expressed in human heart and contributes toaction potential repolarization.The I_Ks channel has at least two components:a majorpore-forming component KCNQ1 channel,and a small auxiliary component KCNE1.When Q1 is expressed alone,it mediates a relatively fast-activating,slowly deactivating Kcurrent.E1 association induces the following changes in the Q1 channel function:increasein single channel conductance and thus a larger macroscopic current amplitude,slowing ofactivation and positive shift in the voltage dependence of activation,and suppression ofinactivation.How E1 modulate the KCNQ1 function haven't fully understood.In the extracellularend of S1 stems from the observations that a short stretch of amino acids here (positions140～147) harbors six arrhythrnia-associated mutations.Furthermore,for at least three ofthem,the manifestation of the abnormal phenotypes requires E1 coexpression.Themutations introduced into the extracellular end of E1 TMD can interfere with the actions ofsome of these I_Ks activators.These observations suggest that the extracellular end of S1 inQ1 is involved in Q1/E1 interactions,which impact on the channel gating kinetics andcurrent amplitude.We use the approach of"disulfide trapping"to probe KCNQ1/KCNE1 interactions onthe extracellular surface.We coexpress Cys-substituted Q1/E1 pairs in COS-7 cells,anduse Western blots to detect disulfide bond formation between Q1 & E1.We then usevoltage clamp experiments in oocytes to probe for functional consequences,as well as thestate dependence and rate of disulfide bond formation. PartⅠThe mutations of KCNQ1 and KCNE1 preserve the Q1channel function or Q1/E1 interactionsIn this study,we replace all native Cys in Q1 with Ala to create the Q1(-Cys)-WTbackground.We then replace Ile at position 145 with Cys to create Q1 145C.We alsointroduce Cys into E1 positions 40～43.We express all three Q1 variants (Q1-WT,Q1(-Cys)-WT,and Q1(-Cys)-I145C) alone or with E1 variants (E1 WT,E1 40C,E1 41C,E142C,and E1 43C) in Xenopus oocytes,and examine their function using the 2-cushionpipette voltage clamp technique.When the Q1 variants are expressed alone,they exhibitvoltage-gated channel function similar to each other.The most noticeable differencebetween Q1-WT and Q1 (-Cys)-WT is a slowing of deactivation in the latter,inconjunction with a negative shift in its voltage dependence of activation.Therefore,replacing all native Cys in Q1 with Ala causes a stabilization of channels in the open versusclosed states.Replacing Ile-145 with Cys in the Q1 (-Cys)-WT background does not inducefurther changes in the channel function.For all of Q1/E1 complexes,the E1 variants consistently induce the following changesin Q1 variants:slowing activation,positive shift in the voltage dependence of activation,and increase in current amplitude.These observations indicate that the mutant Q1/E1 pairs preserve the essential aspectsof the native conformation in the WT Q1/E1 channel complex.Therefore,the informationabout Q1 and E1 interactions obtained from the following disulfide trapping experimentscan be used to infer the proximity relationship in the native conformation of the Q1/E1channel complex. PartⅡThe state dependence interaction between KCNQ1 145and KCNE1 40 or 411.The interaction between the Q1 145 and E1 40 or 411.1 Disulfide bond formation between Q1 145C and E1 40C or E1 41CWe use COS-7 cell expression and immunoblots to test disulfide bond formationbetween Q1 145C and E1 40C～43C.COS-7 cells are transfected with differentcombinations of Q1 and E1 variants and cultured for 48h before experiments.Q1 145Cexpressed alone or coexpressed with E1 variants migrates as a major 60-kD band.When itis coexpressed with E1 40C or E1 41C,an extra prominent 80-kD band appears,sometimesaccompanied by a weaker 75-kD band,the ratios of 80- to 60-kD band intensities are 0.30±0.03 for Q1 145C/E1 40C and 0.12±0.05 for Q1 145C/E1 41C.No such bands are seenwhen Q1 145C is coexpressed with E1-WT,and only extremely faint bands are seen whenQ1 145C is coexpressed with E1 42C or E1 43C.The 80- and 75-kD band sizes are similarto the expected sizes of disulfide-linked Q1 and E1 complexes.DTT treatment abolishesboth 80- and 75-kD bands in the Q1 145C/E1 40C lane.We propose that Cys introducedinto Q1 position 145 can form disulfide bonds with Cys at E1 positions 40 and 41,but notat E1 position 42 or 43.The ratios of 80- to 60-kD band intensities are 0.30±0.03 for Q1145C/E1 40C and 0.12±0.05 for Q1 145C/E1 41C.1.2.The presence of E1 in the 80- and 75-kD Q1-positive bands in the whole cell lysateof Q1 145C/E1-G40CWe have made several attempts using the Alomone E1 pAb to probe for E1-positivebands in the 80-75kD range that are specific for Q1 145C/E1 40C and Q1 145C/E1 41Clanes.No,or only very faint,bands in the expected size range can be detected.We use immunoprecipitation to test whether we can selectively coimmunoprecipitateE1 40C with Q1 145C,but not E1 WT with Q1 145C.E1 WT expressed alone cannot be immunoprecipitated by the V5 mAb,Immunoprecipitate of Q1 145C/E1-WT manifests astrong Q1 band but extremely faint E1 bands.However,DTT-treated immunoprecipitate ofQ1 145C/E1 40C reveals strong E1 bands,accompanied by a strong 60-kD Q1 monomerband but a total disappearance of the 80- and 75-kD Q1 bands.These observations confirmthe presence of E1 in the 80- and 75-kD Q1-positive bands in the whole cell lysate of Q1145C/E1 40C.1.3.The different molecular weight E1 bands are due to different degrees ofglycosylation,and the 80- and 75-kD bands detected in Q1 immunoblot containglycosylated E1The E1 immunoblot exhibits a complex banding pattern.In addition to the expected15-kD unglycosylated E1 band,we see E1 bands at 28,23,20,and 16-kD.To test whetherthese higher molecular weight E1 bands are due to different degrees of glycosylation,andto further test whether the 80- and 75-kD bands detected in Q1 immunoblot containglycosylated E1,we incubate whole cell lysate of Q1 145C/E1 40C with PNGase F for 24or 48 h.We use such long incubation times to ensure as complete deglycosylation aspossible under our experimental conditions.PNGase F treatment collapses the 80-kD bandinto the 75-kD band,suggesting that the former contains glycosylated E1 while the lattercontains unglycosylated E1.In the E1 immunoblot,PNGase F treatment produces a major15-kD band,confirming that this represents unglycosylated E1.2.The impact of the interaction between Q1 145 and E1 40 or 41 on Q1/E1channelfunction2.1.Disulfide bond formation between Q1 145C/ E1 40C stabilize channel in theactivated state.Q1 145C coexpress with E1 40C,oocytes are treated with DTT (10 mM for 10 min,or25 mM for 5 min) and thoroughly rinsed before current recording.Before DTT treatment,Q1 145C/E1 40C manifests a slowly activating current without constitutive component,after DTT treatment,an instantaneous current component appears,so that the activation curve has two components:a constitutive component and a time-dependent component.DTT treatment shifts the activation curve in the negative direction (V_0.5 32.3±4.0 and 2.1±6.3 mV,before and after DTT respectively).We propose that the disulfide bondformation between Q1 145C/E1 40C stabilize channel in the activated state.2.2 Disulfide bond formation between Q1 145C/E1 40C stabilize channel in theresting state.Q1 145C coexpress with E1 41C,oocytes are treated with DTT (10 mM for 10 min,or25 mM for 5 min) and thoroughly rinsed before current recording.Without DTT treatment,the Q1 145C/E1 41C channel manifests an extremely positively shifted activation curve.DTT treatment shifts the activation curve in the negative direction (V_0.5 80.2±5.7 and 55.0±5.8 mV,before and after DTT respectively).We propose that the disulfide bondformation between Q1 145C/E1 41C stabilize channel in the activated state.3.The state dependence of Q1 and E1 interaction3.1 Q1 145C and E1 40C form a disulfide bond when the channel reaches the activatedstateThe membrane voltage is stepped from V h-100 to +60 mV for 2 s and then to -60mV for 2 s once every 60 s.Oocytes expressing Q1 145C/E1 40C without any DTTtreatment exhibit a stable phenotype of slowly activating currents without anyinstantaneous current component.Exposing the oocytes to DTT induces a gradual increasein the current amplitudes.However,in the presence of DTT the current kinetics remains thesame,i.e.,slow activation without any instantaneous component.During DTT washout theoocyte membrane is depolarized to +60 mV for 2 s once every 60 s,the Q1 145C/E1 40Cchannels are repetitively activated.There is a further increase in the total current amplitude.More importantly,an instantaneous current component gradually develops.In another experiment,during DTT washout,the oocyte membrane is held at -100 mVto maintain in the resting state.After thoroughly removing DTT from the bath,pulsing isresumed.The first pulse induces a slowly activating current with a very little instantaneous component.However,a prominent instantaneous current component develops during thesecond pulse and remains relatively stable during the following pulses.We use COS-7 expression and immunoblotting experiment to test whether disulfidebond formation between Q1 145C and E1 40C after DTT washout can also be facilitated bychannel activation.80-kD band in cells coexpressing Q1 145C and E1 40C.Treating theCOS-7 cells with 10 mM DTT for 10 min,followed by DTT washout for 10min using theregular medium (containing 5 mM [K]_o),results in a total disappearance of the 80-kDband.However,when the [K]_o in the washing medium is elevated to 100 mM,and thus theCOS-7 cell membrane is depolarized to 0 mV for 10 min during the washout period,a faintbut definite 80-kD band reappears.Taken together,these data indicate that after DTT washout,145C on Q1 and 40C onE1 can form a disulfide bond in the activated state of the channel.3.2.The rate of disulfide bond formation between Q1 145C and E1 40C is 1200 ms^-1To more accurately estimate the rate of disulfide bond formation between Q1 145Cand E1 40C,after thoroughly removing DTT from the bath solution while preventingdisulfide bond formation by holding the membrane at -100 mV for 15 min,the membrane isstepped to +60 mV for 0.2 s once every 15 s.A constitutive component (Istep) graduallydevelops.The time course of growth of I step is fit with a single- exponential function.Thetime constant is converted to the cumulative channel activation time at +60 mV,generatingaτvalue of 0.85±0.14 s for disulfide bond formation between Q1 145C and E1 40C in theactive state,the rate of disulfide bone formation between the Cys pair is the reciprocal ofthe time constant,the value is 1200 ms^-1.3.3 Q1 145C and E1 41C can form a disulfide bond in the resting state and the rate ofdisulfide bond formation is 2.4 ms^-1Both Q1 position 145 and E1 position 41 are mutated to Cys,the Q1/E1 channelcomplex is uniquely stabilized in the resting state.DTT application induces a gradualincrease in the current amplitude,consistent with an increase in the channel open probability secondary to a reduction of disulfide bonds between Q1 145C and E1 41C.The data indicate that Q1 145C and E1 41C can form a disulfide bond in the resting state.Upon DTT washout,the current amplitude gradually declines.This decline in currentamplitude after DTT removal is consistent with a reformation of disulfide bonds betweenQ1 145C and E1 41C.This time course can be well described by a single exponentialfunction.The time constant is converted to cumulative time at -100 mV,when the Q1145C/E1 41C channels are in the resting state.This gives an estimatedτvalue for disulfidebond formation between Q1 145C and E1 41C in the resting state of 425±86 s.The rate ofdisulfide bond formation between Q1 145C and E1 41C is the reciprocal of the timeconstant,the value is 2.4 ms^-1.Conclusion:The major findings in this study can be summarized as the following.①Cys introduced into Q1 position 145 can form a disulfide bond with Cys introduced into E1positions 40 and 41,but not Cys introduced into E1 position 42 or 43.②Q1 145C/E1 40Cexhibits a constitutive component after DTT treatment.This constitutive component can beattributed to disulfide bond formation between Q1 145C and E1 40C when the channelreaches the activated state,which then stabilizes the channel in the activated state.③Q1145C/E1 41C exhibits an extremely positive voltage range of activation,and DTTtreatment shifts the activation curve in the negative direction.These observations indicatethat Q1 145C and E1 41C can form a disulfide bond in the resting state,which furtherstabilizes the channel in the resting state.Previous studies have shown that E1 can interactwith Q1 S6 and S4 regions.We propose that E1 binds in a"KCNE binding pocket"between VSDs of two adjacent Q1 subunits,and juxtaposed to the pore domain (PD) of athird Q1 subunit.Different E1 positions 40 or 41 come very close to the same Q1 positionwhen channel in the different states.A new concept emerging that E1 is not a stationary,and it can engage in the I_ks gating in a more dynamic fashion.