Reference #3 (Torres AM et al.): S5P
- Alert me when: new articles cite this article Download to Citation Manager All Versions of this Article: 278 / 43 / 42136 (most recent) M212824200v1 Structure of the HERG K + Channel S5P Extracellular Linker ROLE OF AN AMPHIPATHIC alpha -HELIX IN C-TYPE INACTIVATION * (image) Allan M
- Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner
- However , the extracellular loop connecting the pore helix to the top of S5 (S5P loop) in HERG is very different from that in other VGK family members
- First , the S5P loop in HERG is about 40 amino acids long , compared with 10-15 in most other members of the VGK family (9 , 23 ) (also see Fig. 1 )
- Second , many mutations in the S5P loop disrupt the inactivation process in HERG (19 , 23 , 24 )
- Third , toxins that bind to the S5P loop of other VGK channels , e.g. agitoxin and charybdotoxin (25 ) , do not bind to HERG
- The S5P loop of HERG therefore appears to be a critical region of the protein , but at present there is little specific information known about its three-dimensional structure
- In this study we have used a combination of CD spectropolarimetry , two-dimensional 1H NMR spectroscopy , and electrophysiology to investigate the structure and function of the S5P linker of the HERG K+ channel
- Our findings show that the S5P linker contains an amphipathic alpha -helix
- Exogenous application of the S5P peptide fragment or a peptide corresponding to the amphipathic alpha -helix results in altered ionic selectivity and disruption of inactivation of the HERG K+ channel
- These results suggest that the amphipathic alpha -helix in S5P is critical for inactivation of HERG K+ channels
- Three peptides were synthesized in this study: a 42-residue peptide corresponding to the S5P linker of HERG (residues Ala570-Tyr611) , to which we refer as the S5P peptide ; a 42-residue peptide in which the putative amphipathic helix corresponding to residues Gly584-Lys595 (23 ) was replaced with a GGGSGGGSGGGS linker , to which we refer as the del-helix peptide ; and finally a 19-residue peptide corresponding to the putative amphipathic helix and four residues at each end (i.e. Ser581-Ser599 of wild type HERG) , to which we refer as the helix peptide
- The NMR spectroscopy sample was prepared by dissolving 2.6 mg of the S5P peptide in ~400 µl of 90% H2O , 10% D2O (v / v) containing 12 mg of SDS-d25 (~100 mM ; the critical micellar concentration for SDS is 8 mM) in a 5-mm outer diameter susceptibility-matched microcell (Shigemi , Tokyo , Japan)
- Analysis of the ensemble of S5P structures was also carried out using PROMOTIF , a program that identifies structural motifs in proteins (46 )
- Peptide concentration was 0.45 mg / ml for the S5P peptide or 0.15 mg / ml for the del-helix and helix peptides , in 10 mM sodium phosphate , pH 3.0 and 7.0 , with or without the addition of 100 mM SDS
- A , typical examples of current traces (control , thin trace ; with S5P peptide , thick trace) obtained for test pulses of +30 mV (panel a) , -20 mV (panel b) , and -70 mV (panel b , taken from the same cell as used in Fig. 8 )
- The effect of S5P peptide on HERG currents was quantified by fitting single exponential functions (thin lines superimposed on current traces) to the ascending (denoted by open boxes) or descending (denoted by closed box) portions of the currents recorded during application of S5P peptide
- In panel a , the onset of the current increase in the presence of S5P peptide had a time constant of 123 ms , and in panel b , the time constant was 89 ms
- The apparent peptide binding time constant was obtained by dividing the current recorded in the presence of peptide (I S5P) by the control current (I con ; see inset ; also see text for details of method used to deconvolve time constants)
- for the time constants measured from the ascending phase (open symbols , n = 4) and descending phase (after correction for deactivation ; closed symbols , n = 5) of currents following addition of S5P peptide
- C , summary of the maxi mum change (increase at positive potentials (open symbols) or decrease at potentials < = -20 mV (closed symbols)) in current following application of 100 µM S5P peptide (mean ± S.E
- D , typical examples of current traces recorded at -70 , -80 , and -90 mV during control conditions and -60 , -70 , and -80 mV in the presence of S5P peptide
- In this example the reversal potential was shifted from 80 to 64 mV during addition of the S5P peptide
- E , typical examples of currents recorded at +30 mV during application and wash-off of 100 µM S5P peptide (indicated by a thick bar above the voltage protocol)
- Typical examples of the effect of 100 µM S5P peptide applied for 1 s on HERG currents recorded during test potential steps in the range +30 to -110 mV
- The dotted boxes (thin , control ; thick , with S5P peptide) on the voltage protocol indicate which regions are shown in the current traces
- Application of S5P peptide resulted in increased current at depolarized potentials (at least -20 mV) , a more rapid decline in current at intermediate potentials (-30 to -60 mV) , and a reversal of current from outward to inward at -70 and -80 mV
- RESULTS (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES Circular Dichroism Spectropolarimetry—Far-UV CD spectropolarimetry spectra of the S5P HERG peptide in 10 mM sodium phosphate buffer with and without 100 mM SDS at 20 °C and pH 3.0 are shown in Fig. 2
- The large mini mum between 195 and 200 nm and ellipticity close to zero at 222 nm for the S5P peptide in aqueous solution (thin line) indicates that the peptide does not have a well defined secondary structure under these conditions
- This indicated that the S5P peptide contains helical elements in the micellar environment at pH 3.0
- Comparison of the far-UV CD spectropolarimetry spectra of the HERG S5P loop peptide in 10 mM phosphate , pH 3.0 (thin line) , and in 10 mM phosphate , 100 mM SDS , pH 3.0 (thick line)
- A , far-UV CD spectropolarimetry spectra for S5P peptides
- However , in the presence of SDS micelles (thick lines) both the S5P peptide and the helix peptide display elements of helical structure , whereas the del-helix remains unstructured
- Calculation of the mean residue molar ellipticity , which takes into account the number of peptide bonds in the peptide , indicates that the helix peptide is almost completely helical in SDS micelles , with very little of the sequence in alternative conformations , whereas the S5P contains significant random coil content in addition to the helical elements
- This is consistent with the experimentally determined structure of the S5P peptide and what would be expected from secondary structure prediction algorithms
- S5P peptide causes a shift to inward current , the del-helix peptide has minimal effect on the current and the helix peptide has a similar effect to the full-length peptide
- NMR Spectroscopy—The two-dimensional NOESY spectrum of the S5P peptide in aqueous solution at 25 °C had only a few very weak cross-peaks , suggesting that the peptide had a flexible , predominantly random coil structure (data not shown) that was consistent with the CD spectropolarimetry data (Fig. 2 )
- Amide region of 200-ms NOESY spectrum of S5P peptide in 100 mM SDS at pH 3.3 and 30 °C
- Assignments of proton resonances from the S5P peptide were made using standard methods (49 ) of analyzing two-dimensional total correlation spectroscopy and NOESY spectra
- The absence of a group of residues with chemical shift deviations greater than +0.1 ppm indicated that no beta -sheet structure is expected for the S5P HERG peptide
- The rest of the S5P HERG peptide , residues 1-15 and 23-38 , is likely to be flexible or in random conformations
- Summary of NMR spectroscopy data for secondary structure prediction in S5P peptide in 100 mM SDS
- In addition to the presence of significant amide-amide NOE connectivities , the two-dimensional NOESY spectrum of S5P showed many medium range connectivities between residues 14-24 and 38-42 (Fig. 4 )
- No long range NOEs were detected , implying that the S5P peptide does not have a well defined tertiary structure
- Structural Fold—Comparison of the 20 best S5P structures showed that S5P did not have a distinct three-dimensional fold in SDS micelles
- Also , PROMOTIF analysis (46 ) of the ensemble of S5P structures predicts that the regions defined by residues 16-25 and 35-42 form alpha -helices in the presence of SDS micelles
- NMR spectroscopy structure of S5P peptide in 100 mM SDS micelles
- B , the whole S5P peptide showing the helices at the center and the N-terminal end of the molecule
- In the KcsA structure (22 ) , the ends of the extracellular loop that corresponds to S5P in HERG , (i.e. Ala50 and Tyr62 in KcsA) are close together , being separated by only ~11 A
- We attempted to constrain the ends of the S5P peptide by introducing a 'dummy' distance constraint of 10.9 A between the backbone C alpha of Ala1 and that of Tyr42 in the NMR spectroscopy structure calculation
- To investigate whether exogenous application of the S5P peptide would have any effect on full-length HERG channels , we superfused cells with 1 µM S5P peptide , and the membrane was depolarized from -80 to +40 mV for 0.5 s followed by a hyperpolarization to -120 mV for 1 s , and the protocol repeated every 5 s
- Application of the 42-residue S5P peptide caused a reversible suppression of HERG current (Fig. 6 , B and C )
- Application of 1 µM S5P peptide to CHO cells transfected with HEAG or rELK2 channels resulted in no reduction in current (data not shown)
- B , typical example of currents recorded from a CHO-HERG cell using the voltage protocol shown in A before (i) , after 1 min superfusion with 1 µM S5P peptide (ii) , and 6 min after washout of the peptide (iii)
- C , changes in peak tail current recorded at -120 mV prior to superfusion , during superfusion , and following washout of 1 µM S5P peptide
- The thin lines show single exponential fits to the data for the onset of the effect of S5P peptide ({tau} = 17.8 s , {lambda} = 0.056 s-1) and the wash off of the S5P peptide effect ({tau} = 53.7 s , k-1 = 0.019 s-1)
- D , dose dependence of effects of S5P peptide on currents recorded at -120 mV (n = 3-4 at each dose) , using the protocol shown in A
- To investigate further the binding of the S5P peptide to HERG channels , we used a picospritzer to rapidly apply peptides and minimize the amount of peptide used in each experiment
- First we investigated the dose response of the effect of S5P peptide by applying the peptide at doses ranging from 0.1 to 100 µM (Fig. 6D ) using the same voltage protocol as used in Fig. 6A
- This value is similar to that measured from the on and off rates for 1 µM S5P , i.e. 1.1 µM
- To investigate the voltage dependence of the effect of S5P peptide on HERG channels , we used a voltage protocol where within each sweep channels were first activated by stepping to +40 mV for 500 ms and then stepped to a test voltage in the range +30 to -110 mV followed by a -120 mV step for 800 ms to fully deactivate the channels
- Based on the on rate for S5P peptide binding to HERG K + channels calculated above , we used 100 µM S5P peptide in these experiments so as to achieve >90% binding within 1 s of application
- A , a typical example of a family of currents recorded during a double pulse protocol to examine the voltage dependence of S5P peptide effects on HERG channels
- The channels were deactivated by hyperpolarizing the cell to -120 mV for 800 ms , and the protocol was repeated except that during the second test pulse 100 µM S5P peptide was applied for1s(as indicated by the solid bar)
- The voltage protocol is illustrated above the current trace , and the dotted boxes (thin , control ; thick , with S5P peptide) indicate the regions that are plotted
- 100 µM S5P peptide was applied for 1 s as indicated by the thick line in the voltage protocol
- The effect of rapid application of 100 µM S5P peptide was voltage-dependent
- For example , application of S5P peptide caused an increase in outward current at +30 mV (Fig. 7A , solid arrows) but a significant decrease in the current at -50 mV (Fig. 7A , dotted arrows)
- Both the control currents (Fig. 7B , thin line) and currents recorded during application of the S5P peptide (Fig. 7B , thick line) were superimposable
- Also of note from Fig. 7B is that in the presence of the S5P peptide the current recorded at -70 mV reversed from an outward current to a small inward current , suggesting that application of the peptide altered the selectivity of the channel (see below)
- To quantify the effect of the S5P peptide on HERG channels , we analyzed the rates of change in current magnitude following the addition of the peptide (i.e. the apparent on rates ; Fig. 9 , A and B ) , the maxi mum increase (or decrease) in current during the 1-s application of the peptide (Fig. 9 , A and C ) , and the change in reversal potential caused by application of the peptide (Fig. 9D )
- At -20 mV there were two phases to the change in current during application of S5P peptide , i.e. an initial increase in current followed by a slower decrease in current
- In the example illustrated in Fig. 9A (panel c) , the time constant of deactivation in the control trace (no peptide present) was 223 ms , and the time constant of current decline following application of the S5P peptide was 65 ms
- We measured the maxi mum increase (at potentials > = = ' BORDER = '0'> -20 mV) and decrease (at potentials < = -20 mV) in current during application of the S5P peptide from the single exponential curves fitted to the data after correcting for channel deactivation if appropriate (Fig. 9A , panel c , inset)
- The maxi mum change in current during a 1-s application of 100 µM S5P peptide varied significantly with voltage
- The mean change in reversal potential was from -83 ± 1.5 mV (normal Tyrode solution) to -68 ± 2.2 mV (in the presence of S5P peptide , n = 6)
- From the change in reversal potential following application of S5P peptide , we estimate , using Equation 1 , that the permeability ratio for K + over Na + (P K / P Na) was reduced from 188 ± 38 to 27 ± 5 (n = 6)
- In the example illustrated in Fig. 9E , the time constant for the increase in current in the presence of S5P peptide was 123 ms , and the time constant of the decrease in current following wash-off of the peptide was 2.26 s
- The above results , most notably the altered ionic selectivity , suggest that the exogenously applied S5P peptide is interacting either directly with the selectivity filter of the channel or binding to the channel in such a way as to cause a change in the conformation of the selectivity filter
- To determine which region of the S5P peptide was responsible for the interaction with the pore region of the HERG channel , we synthesized two additional peptides
- All three peptides , S5P , del-helix , and helix peptides , adopted a random coil conformation in aqueous solution at pH 7.0 (Fig. 10A )
- In the presence of SDS micelles , the del-helix peptide remained in a random coil conformation , and the S5P peptide contained some alpha -helical elements (the spectra obtained at pH 3.0 (Fig. 2 ) and pH 7.0 (Fig. 10A ) were indistinguishable) , and the helix peptide adopted an almost pure alpha -helix , as expected
- Typical examples of HERG tail currents recorded at -80 mV during control sweeps or during application of the S5P , del-helix , or helix peptides are shown in Fig. 10B
- The helix peptide , however , had a very similar effect to that observed with the full-length S5P peptide (also see below)
- Qualitatively , the effects seen with application of the helix peptide are very similar to those seen with the S5P peptide (compare Figs
- The apparent time constants for peptide binding were faster for the helix peptide than for the S5P peptide (compare Figs
- 9B and 11B ) , the off rate was also slightly faster for the helix (time constant for dissociation at +30 mV was 0.85 ± 0.15 s (n = 5) for the helix peptide compared with 2.06 ± 0.56 s (n = 4) for the S5P peptide) , and the maxi mum increase in current seen at positive potentials was less for the helix peptide than for S5P peptide (Table I )
- However , the shift in the reversal potential (14 ± 2 mV , n = 4) following addition of the helix peptide was similar to that seen with the S5P peptide (16 ± 2 mV , n = 6)
- View this table: [in this window] [in a new window] TABLE I Summary of effects of S5P , helix , and del-helix peptides on HERG current For the S5P and helix peptides , the measurements are from n = 4—6 cells , and for the del-helix peptide , the measurements are from n = 3 cells
- The shift in reversal potential following application of either the 42-residue S5P peptide or the 19-residue helix peptide was consistent with a decreased selectivity for K + over Na +
- In the absence of external Na + , acute application of either the S5P peptide or the helix peptide caused only modest decreases in current at all test potentials (Fig. 12A )
- It should be noted , however , that both the S5P and helix peptides caused inhibition of current at -120 mV when applied for 1 min in NMDG + external solutions (Fig. 12B )
- A , typical examples of HERG tail currents recorded at 0 , -40 , and -80 mV in Na+-free (replaced with NMDG+) solution under control conditions (thin traces) or following addition of 100 µM S5P peptide (thick traces , left-hand panel) or helix peptide (thick traces , right-hand panel)
- B , typical examples of current traces recorded before (thin trace) and after 1 min superfusion with 100 µM S5P or helix peptide (thick traces) in Na +-free (replaced with NMDG +) solution
- DISCUSSION (image)TOP (image)ABSTRACT (image)INTRODUCTION (image)EXPERIMENTAL PROCEDURES (image)RESULTS (image)DISCUSSION (image)REFERENCES The S5P Linker Contains an Amphipathic Helix—In this work we have identified an amphipathic alpha -helix that is present in the extracellular linker connecting the outer ends of S5 and the pore helix of the HERG K+ channel
- It is possible that the presence of the glycine is the reason that the S5P peptide is unstructured in water
- Second , application of exogenous S5P peptide to wild type HERG K+ channels caused dramatic changes in channel properties (see below)
- The Amphipathic Helix Interacts with the Pore Region of the Channel—Exogenous application of the S5P peptide had at least two effects on HERG currents
- Thus it seems that the major site of interaction between the S5P peptide and the rest of the channel involves the amphipathic alpha -helix
- Current Suppression—When cells were exposed for prolonged periods to S5P peptide , current was suppressed in a dose-dependent manner with an IC50 of ~1.9 µM (Fig. 6D )
- This inhibitory effect of the S5P peptide was independent of external Na+ (Fig. 12B ) ; however , in the absence of external sodium , the potency of the peptide appeared to be much reduced
- Disrupted Inactivation and Altered Selectivity—The addition of 100 µM S5P peptide to HERG K + channels caused a depolarizing shift in the reversal potential by 16 ± 2 mV (Fig. 9D and Table I )
- However , when all external Na + was replaced by NMDG + , the S5P peptide no longer caused a shift in reversal potential (Table I )
- If a decrease in selectivity for K + relative to Na + was the only effect of acute application of S5P peptide , then one would not have expected an increase in current at positive voltages (Fig. 8 , panels a-c) , because the positive shift in reversal potential would result in a smaller driving force for outward current flow
- Such a hypothesis is also consistent with previous reports showing that many of the mutations in the S5P linker that affect inactivation also affect the selectivity for K + over Na + (19 )
- Thus we suggest that the exogenously applied S5P peptide either itself directly binds to the pore region and thereby disrupts inactivation and K +-Na + selectivity , or it binds to the outer pore region in such a way that it induces a conformational change in the selectivity filter region
- The very significant reduction in the effect of acute application of S5P peptide when external Na + was replaced by NMDG + (compare Figs
- 9 and 12 ) suggests that the increase in current seen with application of S5P peptide at positive potentials is Na +-dependent
- Thus it may be that the S5P peptide is competing with external Na + for binding to the site that when occupied by Na + promotes inactivation (52 )
- Is Binding of S5P to the HERG K+ Channel Voltage-dependent?—The effect of the acute application of S5P peptide to HERG K+ channels was clearly voltage-dependent (Figs
- The voltage dependence of the time constants for change in current following addition of S5P peptide (Fig. 9B ) also suggests that there are two components to peptide binding
- The augmentation of current at positive potentials but inhibition at negative potentials following application of the S5P peptide (Fig. 9C ) is reminiscent of the effect of Ba2+ on HERG K+ channels (53 ) , i.e. 2 mM Ba2+ blocks HERG currents at negative voltages but increases the current at positive voltages , an effect that has been attributed to a voltage-dependent competition between Ba2+ and Na+ for binding to an outer pore-binding site (52 )
- Although the voltage protocols used in the earlier Ba2+ studies (52 , 53 ) and this study are different , it is possible that the S5P peptide , like Ba2+ , may be competing with Na+ for binding to an outer pore-binding site and that this competition is voltage-dependent
- Comparison of the Effects of the S5P Peptide and the Helix Peptide—The effects of adding the S5P peptide to HERG channels could be reproduced , qualitatively , with a 19-residue peptide containing only the amphipathic helix and four flanking residues on either side
- For example the time constant for binding of 100 µM peptide at 0 mV was 145 ± 36 ms (n = 5) for S5P peptide compared with 36 ± 7ms(n = 3) for the helix peptide (Table I )
- Second , the maxi mum increase in current seen during peptide application at positive potentials was greater for the S5P peptide than for the helix peptide
- For example the maxi mum increase at +30 mV was 56 ± 22% (n = 4) for the S5P peptide and 9 ± 3% (n = 3) for the helix peptide (Figs
- These data suggest that a region of the S5P peptide not present in the helix peptide , for example the charged motif at the C-terminal end , inhibits access of the amphipathic helix to a binding site on the channel
- However , once it has bound the S5P peptide is more effective than the helix peptide at altering channel function
- How Does the S5P Peptide Interact with the Rest of the HERG Channel?—Given that the S5P linker is critical for inactivation of HERG K+ channels and inactivation in HERG K+ channels is voltage-dependent (1 , 10 -12 ) , it is possible that voltage-dependent binding of the S5P linker to the outer pore of the channel could contribute to the voltage-dependent inactivation of HERG K+ channels
- There are a number of charged residues in the S5P linker including an aspartate (Asp591) in the amphipathic alpha -helix (Fig. 1 ) that in theory could contribute to voltage-dependent binding of the S5P linker to the rest of the channel
- We were not able to define a unique three-dimensional fold for the S5P loop of the HERG K+ channel , despite constraining the ends of the peptide to the homologous residues in the KcsA structure (22 )
- To further address this issue will require additional experimental data to help constrain the possible orientations of the S5P linker and to identify the specific site(s) of interaction between the S5P linker and the rest of the channel
- We have also shown that the S5P peptide is able to interact with the rest of the HERG channel , resulting in suppression of current , an altered selectivity for K + over Na + , and partial disruption of inactivation
- Furthermore , we have shown that it is the amphipathic alpha -helix region that is critical for the effects on ion selectivity and inactivation , because when the amphipathic alpha -helix region of the S5P peptide is replaced with a random coil linker , the peptide no longer affects ion selectivity or inactivation (Fig. 10B and Table I ) , whereas a peptide containing only the helix region has very similar effects on the HERG channels as the full-length peptide (compare Figs
- Interestingly , the S5P peptide and the helix peptide are able to affect HERG channels despite being applied in normal Tyrode solution where in isolation they would be expected to have a random coil conformation
|
2005, KChannelDB.
cmbi.ru.nl), 17-Aug-2005