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Rescue of Aberrant Gating by a Genetically Encoded PAS (Per-Arnt-Sim) Domain in Several Long QT Syndrome Mutant Human Ether-á-go-go-related Gene Potassium Channels*

  • Elena C. Gianulis
    Affiliations
    Program in Molecular Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201

    Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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  • Matthew C. Trudeau
    Correspondence
    To whom correspondence should be addressed: 660 W. Redwood St., HH502, Baltimore, MD 21201. Tel.: 410-706-5551; Fax: 410-706-8341
    Affiliations
    Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants HL-083121 (to M. C. T.) and T32-GM 008181 (to E. C. G.) and a gift from the Helen Pumphrey Denit Trust.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.
Open AccessPublished:May 02, 2011DOI:https://doi.org/10.1074/jbc.M110.205948
      Congenital long QT syndrome 2 (LQT2) is caused by loss-of-function mutations in the human ether-á-go-go-related gene (hERG) voltage-gated potassium (K+) channel. hERG channels have slow deactivation kinetics that are regulated by an N-terminal Per-Arnt-Sim (PAS) domain. Only a small percentage of hERG channels containing PAS domain LQT2 mutations (hERG PAS-LQT2) have been characterized in mammalian cells, so the functional effect of these mutations is unclear. We investigated 11 hERG PAS-LQT2 channels in HEK293 cells and report a diversity of functional defects. Most hERG PAS-LQT2 channels formed functional channels at the plasma membrane, as measured by whole cell patch clamp recordings and cell surface biotinylation. Mutations located on one face of the PAS domain (K28E, F29L, N33T, R56Q, and M124R) caused defective channel gating, including faster deactivation kinetics and less steady-state inactivation. Conversely, the other mutations caused no measurable differences in channel gating (G53R, H70R, and A78P) or no measurable currents (Y43C, C66G, and L86R). We used a genetically encoded hERG PAS domain (NPAS) to examine whether channel dysfunction could be corrected. We found that NPAS fully restored wild-type-like deactivation kinetics and steady-state inactivation to the hERG PAS-LQT2 channels. Additionally, NPAS rescued aberrant currents in hERG R56Q channels during a dynamic ramp voltage clamp. Thus, our results reveal a putative “gating face” in the PAS domain where mutations within this region form functional channels with altered gating properties, and we show that NPAS is a general means for rescuing aberrant gating in hERG LQT2 mutant channels and may be a potential biological therapeutic.

      Introduction

      Congenital Long QT Syndrome (LQTS)
      The abbreviations used are: LQTS
      long QT syndrome
      hERG
      human ether-a-go-go-related gene
      IKr
      rapid delayed-rectifier potassium current
      PAS
      Per-Arnt-Sim
      ANOVA
      analysis of variance.
      is a disorder of the electrical system of the heart characterized by delayed cardiac repolarization that can lead to arrhythmias, syncope, and sudden death (
      • Moss A.J.
      • Schwartz P.J.
      • Crampton R.S.
      • Tzivoni D.
      • Locati E.H.
      • MacCluer J.
      • Hall W.J.
      • Weitkamp L.
      • Vincent G.M.
      • Garson Jr., A.
      • et al.
      ). Type 2 LQTS (LQT2) is caused by genetic mutations in the human ether-á-go-go-related gene (hERG) (
      • Warmke J.W.
      • Ganetzky B.
      ,
      • Trudeau M.C.
      • Warmke J.W.
      • Ganetzky B.
      • Robertson G.A.
      ,
      • Sanguinetti M.C.
      • Jiang C.
      • Curran M.E.
      • Keating M.T.
      ,
      • Curran M.E.
      • Splawski I.
      • Timothy K.W.
      • Vincent G.M.
      • Green E.D.
      • Keating M.T.
      ). hERG encodes the major subunit of the rapidly activating delayed-rectifier potassium current (IKr), which plays an essential role in the final repolarization of the ventricular action potential (
      • Trudeau M.C.
      • Warmke J.W.
      • Ganetzky B.
      • Robertson G.A.
      ,
      • Sanguinetti M.C.
      • Jiang C.
      • Curran M.E.
      • Keating M.T.
      ). hERG exhibits characteristic slow closing (deactivation) kinetics that are regulated by an N-terminal Per-Arnt-Sim (PAS) domain, which help to specialize the channels for their role in the heart (
      • Trudeau M.C.
      • Warmke J.W.
      • Ganetzky B.
      • Robertson G.A.
      ,
      • Morais Cabral J.H.
      • Lee A.
      • Cohen S.L.
      • Chait B.T.
      • Li M.
      • Mackinnon R.
      ,
      • Wang J.
      • Trudeau M.C.
      • Zappia A.M.
      • Robertson G.A.
      ,
      • Viloria C.G.
      • Barros F.
      • Giráldez T.
      • Gómez-Varela D.
      • de la Peña P.
      ,
      • Wang J.
      • Myers C.D.
      • Robertson G.A.
      ,
      • Sale H.
      • Wang J.
      • O'Hara T.J.
      • Tester D.J.
      • Phartiyal P.
      • He J.Q.
      • Rudy Y.
      • Ackerman M.J.
      • Robertson G.A.
      ).
      Loss of hERG function, and thus, loss of IKr (
      • Brunner M.
      • Peng X.
      • Liu G.X.
      • Ren X.Q.
      • Ziv O.
      • Choi B.R.
      • Mathur R.
      • Hajjiri M.
      • Odening K.E.
      • Steinberg E.
      • Folco E.J.
      • Pringa E.
      • Centracchio J.
      • Macharzina R.R.
      • Donahay T.
      • Schofield L.
      • Rana N.
      • Kirk M.
      • Mitchell G.F.
      • Poppas A.
      • Zehender M.
      • Koren G.
      ), can occur through a number of mechanisms, including defects in channel opening and closing (gating), ion permeation, or protein trafficking (
      • Zhou Z.
      • Gong Q.
      • Epstein M.L.
      • January C.T.
      ). hERG channels containing LQT2 mutations in the PAS domain (hERG PAS-LQT2) exhibit robust currents when studied in Xenopus oocytes (
      • Morais Cabral J.H.
      • Lee A.
      • Cohen S.L.
      • Chait B.T.
      • Li M.
      • Mackinnon R.
      ,
      • Al-Owais M.
      • Bracey K.
      • Wray D.
      ,
      • Chen J.
      • Zou A.
      • Splawski I.
      • Keating M.T.
      • Sanguinetti M.C.
      ,
      • Gustina A.S.
      • Trudeau M.C.
      ); however, most channels with LQT2 mutations located outside the PAS domain do not have measurable currents and show defects in maturation and trafficking when studied in mammalian cells (
      • Zhou Z.
      • Gong Q.
      • Epstein M.L.
      • January C.T.
      ,
      • Furutani M.
      • Trudeau M.C.
      • Hagiwara N.
      • Seki A.
      • Gong Q.
      • Zhou Z.
      • Imamura S.
      • Nagashima H.
      • Kasanuki H.
      • Takao A.
      • Momma K.
      • January C.T.
      • Robertson G.A.
      • Matsuoka R.
      ,
      • Ficker E.
      • Dennis A.T.
      • Obejero-Paz C.A.
      • Castaldo P.
      • Taglialatela M.
      • Brown A.M.
      ,
      • Kagan A.
      • Yu Z.
      • Fishman G.I.
      • McDonald T.V.
      ,
      • Roti Roti E.C.
      • Myers C.D.
      • Ayers R.A.
      • Boatman D.E.
      • Delfosse S.A.
      • Chan E.K.
      • Ackerman M.J.
      • January C.T.
      • Robertson G.A.
      ,
      • Anderson C.L.
      • Delisle B.P.
      • Anson B.D.
      • Kilby J.A.
      • Will M.L.
      • Tester D.J.
      • Gong Q.
      • Zhou Z.
      • Ackerman M.J.
      • January C.T.
      ,
      • Paulussen A.
      • Raes A.
      • Matthijs G.
      • Snyders D.J.
      • Cohen N.
      • Aerssens J.
      ). As only 5 hERG PAS-LQT2 channels have been functionally characterized in mammalian cells (
      • Anderson C.L.
      • Delisle B.P.
      • Anson B.D.
      • Kilby J.A.
      • Will M.L.
      • Tester D.J.
      • Gong Q.
      • Zhou Z.
      • Ackerman M.J.
      • January C.T.
      ,
      • Paulussen A.
      • Raes A.
      • Matthijs G.
      • Snyders D.J.
      • Cohen N.
      • Aerssens J.
      ,
      • Rossenbacker T.
      • Mubagwa K.
      • Jongbloed R.J.
      • Vereecke J.
      • Devriendt K.
      • Gewillig M.
      • Carmeliet E.
      • Collen D.
      • Heidbüchel H.
      • Carmeliet P.
      ,
      • Shushi L.
      • Kerem B.
      • Goldmit M.
      • Peretz A.
      • Attali B.
      • Medina A.
      • Towbin J.A.
      • Kurokawa J.
      • Kass R.S.
      • Benhorin J.
      ,
      • Berecki G.
      • Zegers J.G.
      • Verkerk A.O.
      • Bhuiyan Z.A.
      • de Jonge B.
      • Veldkamp M.W.
      • Wilders R.
      • van Ginneken A.C.
      ), the mechanism for how PAS domain mutations disrupt hERG function when expressed in more physiological conditions remains unclear.
      Previously, we showed that slow deactivation could be restored in LQT2 mutant hERG R56Q channels by application of a genetically encoded PAS domain (NPAS) in Xenopus oocytes (
      • Gustina A.S.
      • Trudeau M.C.
      ). Here, we sought to determine whether NPAS was a general mechanism for rescue of LQT2 mutant channels. To carry out this goal we investigated 1) whether 11 different hERG PAS-LQT2 mutations that were gating deficient in Xenopus oocytes resulted in a loss-of-function in a human heterologous expression system and 2) whether NPAS could restore gating in several different hERG PAS-LQT2 mutant channels with gating defects in a mammalian system.
      We found that the 11 hERG PAS-LQT2 channels exhibited a spectrum of deficiencies in mammalian cells, and only channels with mutations located on one face of the PAS domain were gating deficient. These mutant channels exhibited an array of gating defects, including faster deactivation kinetics and a right-shift in the steady-state inactivation relationship, the combination of which resulted in aberrant currents in response to a dynamic ramp clamp. We found that NPAS rescued gating defects in hERG PAS-LQT2 channels by inducing slower deactivation kinetics and a left-shift in the steady-state inactivation relationship, which restored wild-type-like currents during the dynamic ramp clamp. Thus, NPAS restored function to channels that had a variety of gating defects. Therefore, in this study, we identify a putative “gating face” within the PAS domain, as well as present a general means for rescuing gating-deficient mutant hERG PAS-LQT2 channels.

      DISCUSSION

      In this study, we investigated hERG channels bearing LQT2 mutations in the PAS domain (hERG PAS-LQT2) in a mammalian expression system. Our findings demonstrated that hERG PAS-LQT2 channels exhibited a spectrum of biochemical and functional defects (summarized in Table 2). Most hERG PAS-LQT2 channels (K28E, F29L, N33T, G53R, R56Q, H70R, A78P, and M124R) formed functional ion channels at the cell surface because they exhibited neutravidin-purified biotinylated bands on a Western blot and ionic currents as measured with whole cell patch clamp recordings. Two of the mutant channels (Y43C and L86R) did not have measurable ionic currents or measurable neutravidin-purified biotinylated bands. Instead, these channels had a band on a Western blot of whole cell lysates that corresponded to the immature form of hERG, indicating that these mutant channels most likely had defects in protein maturation. One mutant channel (C66G) had no measurable ionic current and only a faint mature band on a Western blot, but did show an immature band on blots of neutravidin-purified biotinylated samples. We propose that the immature form of hERG may be localized at the plasma membrane in these conditions but that these channels are non-functional. Our data with hERG C66G and L86R are different from previous reports from oocytes that showed robust currents (
      • Chen J.
      • Zou A.
      • Splawski I.
      • Keating M.T.
      • Sanguinetti M.C.
      ). The most likely explanation for differences in channel expression between oocytes (previous study) and HEK293 cells (this study) is a temperature-sensitive folding defect that is apparent with differences in culture temperature for oocytes (16 °C) versus HEK293 cells (37 °C), as described for other hERG LQT2 mutant channels (
      • Furutani M.
      • Trudeau M.C.
      • Hagiwara N.
      • Seki A.
      • Gong Q.
      • Zhou Z.
      • Imamura S.
      • Nagashima H.
      • Kasanuki H.
      • Takao A.
      • Momma K.
      • January C.T.
      • Robertson G.A.
      • Matsuoka R.
      ,
      • Anderson C.L.
      • Delisle B.P.
      • Anson B.D.
      • Kilby J.A.
      • Will M.L.
      • Tester D.J.
      • Gong Q.
      • Zhou Z.
      • Ackerman M.J.
      • January C.T.
      ,
      • Paulussen A.
      • Raes A.
      • Matthijs G.
      • Snyders D.J.
      • Cohen N.
      • Aerssens J.
      ,
      • Zhou Z.
      • Gong Q.
      • January C.T.
      ).
      TABLE 2Summary of expression of hERG PAS-LQT2 channels and functional rescue by NPAS in HEK293 cells
      LQT2BiogenesisSurface biotinylationIonic currentAltered kineticsRescue by NPASGating face
      ImmatureMature
      WT+++
      + indicates the degree of detection of the tested property.
      +++Y
      Y = yes; N = no.
      YNn/a
      n/a, not applicable.
      n/a
      K28E++++YYYYY
      F29L++++YYYNY
      N33T++++++YYYYY
      Y43C+
      Minus indicates an absence of the tested property.
      NNn/an/aY
      G53R++++YYNn/aN
      R56Q+++++YYYYY
      C66G++++YNn/an/aN
      H70R+++++YYNn/aN
      A78P+++++YYNn/aN
      L86R+++NNn/an/aN
      M124R++++YYYYY
      a + indicates the degree of detection of the tested property.
      b Y = yes; N = no.
      c n/a, not applicable.
      d Minus indicates an absence of the tested property.
      Here we found that 5 hERG-PAS-LQT2 mutant channels (K28E, F29L, N33T, R56Q, and M124R) exhibited altered gating, including accelerated deactivation kinetics, a positive, rightward shift in the steady-state inactivation curve, and increased steady-state channel availability compared with WT hERG. The largest differences in gating and steady-state properties were seen in hERG R56Q. Also, we found that the resurgent hERG R56Q current was larger during depolarization than WT hERG but was smaller during repolarization, especially at voltages less than −60 mV compared with WT hERG (see Fig. 6E). We propose that the shift in time of the resurgent peak current and the smaller resurgent current at hyperpolarized voltages in hERG R56Q (see Fig. 6E) may be proarrhythmic.
      As the hERG PAS domain is a highly ordered structure, we wanted to know if there was a relationship between the functional defect and the specific location of each LQT2 mutation. We noticed that the LQT2 mutations that altered gating were situated on the same face of the PAS domain (
      • Morais Cabral J.H.
      • Lee A.
      • Cohen S.L.
      • Chait B.T.
      • Li M.
      • Mackinnon R.
      ) (Fig. 7A); in contrast, the mutations that either had no effect on kinetics or abolished currents lie elsewhere on the PAS domain. The lone exception to this rule was Y43C, which was also located on the common face but exhibited defects in channel biogenesis. Our data are in agreement with previous work performed with Xenopus oocytes in which LQT2 mutations (
      • Al-Owais M.
      • Bracey K.
      • Wray D.
      ,
      • Chen J.
      • Zou A.
      • Splawski I.
      • Keating M.T.
      • Sanguinetti M.C.
      ) or alanine mutations (
      • Morais Cabral J.H.
      • Lee A.
      • Cohen S.L.
      • Chait B.T.
      • Li M.
      • Mackinnon R.
      ) that affected deactivation were located within a common region of the PAS domain. Previous studies described a hydrophobic patch in the PAS domain, within which lies Phe-29 and Tyr-43, where mutations located in this region caused an increase in the kinetics of deactivation (
      • Morais Cabral J.H.
      • Lee A.
      • Cohen S.L.
      • Chait B.T.
      • Li M.
      • Mackinnon R.
      ). However, because non-hydrophobic sites, especially Arg-56 (
      • Gustina A.S.
      • Trudeau M.C.
      ), strongly regulate deactivation, we propose that both hydrophobic and non-hydrophobic residues located nearby one another make up a putative gating face on the PAS domain (Fig. 7A), which regulates deactivation and inactivation either allosterically or by forming a direct interaction with another part of the channel, perhaps the C-terminal region (
      • Gustina A.S.
      • Trudeau M.C.
      ).
      Figure thumbnail gr7
      FIGURE 7PAS crystal structure and schematic of proposed mechanism of NPAS rescue. A, crystal structure of the hERG PAS domain (
      • Morais Cabral J.H.
      • Lee A.
      • Cohen S.L.
      • Chait B.T.
      • Li M.
      • Mackinnon R.
      ). Residues marked in red are all situated on the putative gating face, and LQT2 mutations at these sites, with the exception of Y43C, alter deactivation. B, hERG PAS-LQT2 channels that are gating deficient are rescued by NPAS (yellow) through replacement of the covalently attached, mutated (red dot) PAS domain.
      Understanding the molecular defect of each hERG LQT2 channel gives insight into potential mechanisms for restoring WT-like function to mutant channels. To this end, we tested whether a genetically encoded hERG PAS domain (NPAS) (
      • Gustina A.S.
      • Trudeau M.C.
      ) was a general tool to rescue hERG PAS-LQT2 channels in HEK293 cells. We found that NPAS specifically slowed and restored aberrant deactivation gating in hERG PAS-LQT2 channels. We also found that NPAS specifically left-shifted and restored steady-state inactivation gating in the mutant channels and also specifically left-shifted and restored the steady-state activation curve of hERG N33T. NPAS also decreased and restored steady-state channel availability in hERG N33T and R56Q (see Fig. 5C). Notably, NPAS fully rescued the defective resurgent current properties of hERG R56Q in response to a dynamic ramp clamp. Our findings demonstrate that NPAS is a general means for rescuing gating-defective hERG LQT2 mutant channels.
      In a model we propose to explain the rescue of gating properties by NPAS shown here (Fig. 7B), hERG PAS-LQT2 channels exhibit altered deactivation kinetics and steady-state inactivation properties because critical protein interactions between the PAS domain and the channel were disrupted. NPAS physically interacts with the channel and supplants the covalently attached, mutated PAS domain, thus restoring WT-like deactivation kinetics and steady-state inactivation properties. Recently, the notion of using biological alternatives, such as gene therapy, as treatment options for arrhythmogenic diseases like LQT2 was explored (
      • Cho H.C.
      • Marbán E.
      ). Our findings show that NPAS is a useful tool for rescuing gating-deficient mutant hERG channels, and we propose that NPAS may be a potential biological therapeutic for LQT2.

      Acknowledgments

      We thank Dr. M. Sanguinetti for the hERG PAS-LQT2 cDNAs, Dr. G. Robertson for the anti-hERG-KA antibody, Dr. H. Misono for technical assistance, and Dr. A. Meredith, Dr. J. Montgomery, and A. Gustina for helpful discussions.

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