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Mutations in KCNE1 in long QT syndrome (LQTS): Insights into mechanism of LQTS and drug sensitivity?
EDITORIAL COMMENTARY
Mutations in KCNE1 in long QT syndrome (LQTS): Insights into mechanism of LQTS and drug sensitivity? Matteo Vatta, PhD,* Jeffrey A. Towbin, MD*† From the *Department of Pediatrics (Cardiology) and †Molecular Human Genetics, Baylor College of Medicine, Texas Children’s Hospital, Houston, Texas. In this issue of Heart Rhythm, Wu and colleagues1 describe a mutation in KCNE1, the 129 amino acid single membrane-spanning peptide -subunit that associates with the voltage-gated KCNQ1 channel to form the slow delayed rectifier channel (IKs) in the heart, which causes the long QT syndrome type 5 (LQT5). This mutation, Y81C, is located in the post-transmembrane domain region of the KCNE1 (minK) channel in close proximity to three other LQT5 mutations (S74L, D76N, and W87R). Previously, Lai et al2 described this same mutation in a patient with compound heterozygous KCNE1 mutations (Y81C, K70N). In the report by Wu et al,1 the authors examined the effects of the Y81C mutation on the function and drug sensitivity of IKs channels and provide an interesting experimental approach and potentially novel insight into IKs structure-function correlations and future therapeutic options. KCNE1 interacts with the KCNQ1 channel ␣ subunit via its transmembrane domain, which adopts an ␣-helical structure and interacts with the S6 helix in the pore domain of KCNQ1.3-5 In addition, it is speculated that the cytoplasmic region of KCNE1, a conserved region localized 20 –25 amino acids distal to the transmembrane helix, also plays a role in channel conductance and the function of the KCNQ1/KCNE1 complex responsible for IKs. Typically, KCNE1 increases KCNQ1 single-channel conductance, which leads to an increase in the macroscopic current amplitude, shifts the voltage dependence of KCNQ1 channel activation in the positive direction, and slows its activation. As a result of this regulation, a slow buildup of IKs occurs and results in repolarization abnormalities that translate into prolongation of the QT interval when loss-of-function mutations occur or in pathologic shortening of the QT interval when gain-of-function mutations occur. In the former, LQTS (LQT1 when KCNQ1 is mutated, LQT5 when KCNE1 is mutated) occurs,6 while the relatively newly described short QT syndrome or familial atrial fibrillation occurs in the latter instance.6 Y81C is speculated to cause an LQT5 phenotype, and the authors describe the detailed mechanism that likely plays a role in generating this clinical phenotype. Address reprint requests and correspondence: Jeffrey A. Towbin, M.D., Professor and Chief, Pediatric Cardiology, Baylor College of Medicine, Texas Children’s Hospital, 6621 Fannin St., MC 19345-C, Houston, Texas 77030. E-mail address: [email protected].
Using a comprehensive, broad-based scientific approach employing molecular biologic techniques including sitedirected mutagenesis, biophysical methods including voltage clamp recordings of channel function in oocytes and COS-7 cells, and biochemical studies including biotinylation analysis, immunoblotting, and protein structural analysis, the authors show that the Y81C mutation abolishes the ability of KCNE1 to increase current amplitude through the KCNQ1 channel pore while accentuating the ability of KCNE1 to shift the voltage dependence of KCNQ1 activation in the positive direction. This results in the channel becoming more reluctant to open. The mutation causes these changes genetically in a dominant-negative manner irrespective of cell type studied (oocyte vs. mammalian cells). This, in part, explains the clinical phenotype, which is consistent with LQT5 (a loss of IKs function). Biochemically, the authors provide compelling evidence that the cytoplasmic domain of KCNE1 plays a modulating role in regulating KCNQ1 function via its protein secondary and tertiary structure. A wide variety of conservative mutations in this region in addition to Y81-specific mutations (S68T, K70R, K70Q, E72D, H73K, D76N, Y81F, V109I) disrupt the IKs current, and all four of the previously reported LQT5-related mutations in the post-transmembrane domain region shift voltage dependence of channel activation in the positive direction, which leads to the inference that this region interacts with the gating apparatus of KCNQ1. Since no changes were found in the quantity of the KCNQ1 channel protein at the cell surface (i.e., no evidence of trafficking abnormalities or reduction in channel formation), it is likely that structural changes in KCNE1 result in disrupted protein-protein interactions leading to channel and current dysfunction. Protein sequence prediction software studies strongly support the concept of secondary and tertiary structure rearrangements in Y81C. However, the structural analysis performed by the authors employed software that generates protein models based on direct or comparative x-ray crystallography modeling data and has potential limitations when inferring functional consequences of de novo mutations. However, we believe that the use of protein-protein and protein-environment analyses is a move in the right direction in this form of disease analysis. Previously, scientists have been satisfied with genetic studies and biophysical analysis in defining the “mechanism” of disease. Delisle and colleagues8 were the first to study channel
1547-5271/$ -see front matter © 2006 Heart Rhythm Society. All rights reserved.
doi:10.1016/j.hrthm.2006.07.020
1042 structure-function in their reports of protein trafficking abnormalities associated with potassium channel–induced LQTS. This study extends further the use of a detailed biochemical and protein structural analysis to provide more in-depth knowledge of the mechanism. The work by Wu et al1 also adds novel concepts to therapeutic considerations. Taking into account the global concept that loss of function of IKs results in LQTS, while gain of function abnormalities cause short QT syndrome (SQTS), the authors consider the potential utility of drugs shown experimentally to act as IKs activators (to treat lossof-function mutations that cause LQTS) and suppressors (to treat gain-of-function mutations that cause SQTS). The idea, which is to some degree borne from the work on LQT3 by Schwartz and colleagues9 of the Long QT Registry. In that study, in which a gain-of-function sodium channel defect was treated with mexilitine, a sodium channel blocking agent, the treatment resulted in a shortening of the QT interval (to normal) and correction of the biophysical and electrocardiographic abnormalities.9 Using niflumic acid, an IKs activator, the authors showed that this drug can increase the current amplitude and shift the voltage dependence of activation in the negative direction in KCNQ1 and KCNQ1-KCNE1 wild-type channels and that the effects are amplified by the Y81C mutant. It is interesting to also note in this report that the IKs inhibitors HMR1556 and azimilide have differential effects on both wild-type channels and the Y81C mutant. In fact, the Y81C mutant reduced the effect of the IKs inhibitor HMR1556, while it enhanced the effect of the inhibitor azimilide. This observation supports the speculation that this mutation sensitizes the channel to drug response. Clearly, further work is needed to decipher the role of the functional nonsynonymous ion channel variants associated
Heart Rhythm, Vol 3, No 9, September 2006 with arrhythmogenesis for their ability to modulate the response to various antiarrhythmic drugs. This work suggests that drug effectiveness may rely on specific mutations, the biochemical (protein) alterations, and the position of the mutation within the channel. These studies may help to explain the biochemical and biophysical basis of subjective drug response and the paradoxical effects of drugs, which have profound implications for future management of patients including the potential for customized therapy.7
References 1.
2.
3. 4. 5.
6.
7.
8. 9.
Wu D-M, Lai L-P, Zhang M, Wang H-L, Jiang M, Liu X-S, Tseng G-N. Characterization of an LQT5-related mutation in KCNE1, Y81C: Implications for a role of KCNE1 cytoplasmic domain in IKs channel function. Heart Rhythm 2006;3:1031–1040. Lai LP, Su YN, Hsieh FJ, Chiang FT, Juang JM, Liu YB, Ho YL, Chen WJ, Yeh SJ, Wang CC, Ko YL, Wu TJ, Ueng KC, Lei MH, Tsao HM, Chen SA, Lin TK, Wu MH, Lo HM, Huang SK, Lin JL. Denaturing high-performance liquid chromatography screening of the long QT syndrome-related cardiac sodium and potassium channel genes and identification of novel mutations and single nucleotide polymorphisms. J Hum Genet 2005;50:490 – 496. Tapper AR, George AL Jr. MinK subdomains that mediate modulation of and association with KvLQT1. J Gen Physiol 2000;116:379 –390. Tapper AR, George AL Jr. Location and orientation of minK within the I(Ks) potassium channel complex. J Biol Chem 2001;276:38249 –38254. Melman YF, Krummerman A, McDonald TV. KCNE regulation of KvLQT1 channels: structure-function correlates. Trends Cardiovasc Med 2002;12:182– 187. Splawski I, Tristani-Firouzi M, Lehmann MH, Sanguinetti MC, Keating MT. Mutations in the hminK gene cause long QT syndrome and suppress IKs function. Nat Genet 1997;17:338 –340. Hong K, Piper DR, Diaz-Valdecantos A, Brugada J, Oliva A, Burashnikov E, Santos-de-Soto J, Grueso-Montero J, Diaz-Enfante E, Brugada P, Sachse F, Sanguinetti MC, Brugada R. De novo KCNQ1 mutation responsible for atrial fibrillation and short QT syndrome in utero. Cardiovasc Res 2005;68:433– 440. Delisle BP, Anson BD, Rajamani S, January CT. Biology of cardiac arrhythmias: ion channel protein trafficking. Circ Res 2004;94:1418 –1428. Schwartz PJ, Priori SG, Locati EH, Napolitano C, Cantu F, Towbin JA, Keating MT, Hammoude H, Brown AM, Chen LS. Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na⫹ channel blockade and to increases in heart rate. Implications for gene-specific therapy. Circulation 1995;92:3381–3386.
Mutations in KCNE1 in long QT syndrome (LQTS): Insights into mechanism of LQTS and drug sensitivity? Matteo Vatta, PhD,* Jeffrey A. Towbin, MD*† From the *Department of Pediatrics (Cardiology) and †Molecular Human Genetics, Baylor College of Medicine, Texas Children’s Hospital, Houston, Texas. In this issue of Heart Rhythm, Wu and colleagues1 describe a mutation in KCNE1, the 129 amino acid single membrane-spanning peptide -subunit that associates with the voltage-gated KCNQ1 channel to form the slow delayed rectifier channel (IKs) in the heart, which causes the long QT syndrome type 5 (LQT5). This mutation, Y81C, is located in the post-transmembrane domain region of the KCNE1 (minK) channel in close proximity to three other LQT5 mutations (S74L, D76N, and W87R). Previously, Lai et al2 described this same mutation in a patient with compound heterozygous KCNE1 mutations (Y81C, K70N). In the report by Wu et al,1 the authors examined the effects of the Y81C mutation on the function and drug sensitivity of IKs channels and provide an interesting experimental approach and potentially novel insight into IKs structure-function correlations and future therapeutic options. KCNE1 interacts with the KCNQ1 channel ␣ subunit via its transmembrane domain, which adopts an ␣-helical structure and interacts with the S6 helix in the pore domain of KCNQ1.3-5 In addition, it is speculated that the cytoplasmic region of KCNE1, a conserved region localized 20 –25 amino acids distal to the transmembrane helix, also plays a role in channel conductance and the function of the KCNQ1/KCNE1 complex responsible for IKs. Typically, KCNE1 increases KCNQ1 single-channel conductance, which leads to an increase in the macroscopic current amplitude, shifts the voltage dependence of KCNQ1 channel activation in the positive direction, and slows its activation. As a result of this regulation, a slow buildup of IKs occurs and results in repolarization abnormalities that translate into prolongation of the QT interval when loss-of-function mutations occur or in pathologic shortening of the QT interval when gain-of-function mutations occur. In the former, LQTS (LQT1 when KCNQ1 is mutated, LQT5 when KCNE1 is mutated) occurs,6 while the relatively newly described short QT syndrome or familial atrial fibrillation occurs in the latter instance.6 Y81C is speculated to cause an LQT5 phenotype, and the authors describe the detailed mechanism that likely plays a role in generating this clinical phenotype. Address reprint requests and correspondence: Jeffrey A. Towbin, M.D., Professor and Chief, Pediatric Cardiology, Baylor College of Medicine, Texas Children’s Hospital, 6621 Fannin St., MC 19345-C, Houston, Texas 77030. E-mail address: [email protected].
Using a comprehensive, broad-based scientific approach employing molecular biologic techniques including sitedirected mutagenesis, biophysical methods including voltage clamp recordings of channel function in oocytes and COS-7 cells, and biochemical studies including biotinylation analysis, immunoblotting, and protein structural analysis, the authors show that the Y81C mutation abolishes the ability of KCNE1 to increase current amplitude through the KCNQ1 channel pore while accentuating the ability of KCNE1 to shift the voltage dependence of KCNQ1 activation in the positive direction. This results in the channel becoming more reluctant to open. The mutation causes these changes genetically in a dominant-negative manner irrespective of cell type studied (oocyte vs. mammalian cells). This, in part, explains the clinical phenotype, which is consistent with LQT5 (a loss of IKs function). Biochemically, the authors provide compelling evidence that the cytoplasmic domain of KCNE1 plays a modulating role in regulating KCNQ1 function via its protein secondary and tertiary structure. A wide variety of conservative mutations in this region in addition to Y81-specific mutations (S68T, K70R, K70Q, E72D, H73K, D76N, Y81F, V109I) disrupt the IKs current, and all four of the previously reported LQT5-related mutations in the post-transmembrane domain region shift voltage dependence of channel activation in the positive direction, which leads to the inference that this region interacts with the gating apparatus of KCNQ1. Since no changes were found in the quantity of the KCNQ1 channel protein at the cell surface (i.e., no evidence of trafficking abnormalities or reduction in channel formation), it is likely that structural changes in KCNE1 result in disrupted protein-protein interactions leading to channel and current dysfunction. Protein sequence prediction software studies strongly support the concept of secondary and tertiary structure rearrangements in Y81C. However, the structural analysis performed by the authors employed software that generates protein models based on direct or comparative x-ray crystallography modeling data and has potential limitations when inferring functional consequences of de novo mutations. However, we believe that the use of protein-protein and protein-environment analyses is a move in the right direction in this form of disease analysis. Previously, scientists have been satisfied with genetic studies and biophysical analysis in defining the “mechanism” of disease. Delisle and colleagues8 were the first to study channel
1547-5271/$ -see front matter © 2006 Heart Rhythm Society. All rights reserved.
doi:10.1016/j.hrthm.2006.07.020
1042 structure-function in their reports of protein trafficking abnormalities associated with potassium channel–induced LQTS. This study extends further the use of a detailed biochemical and protein structural analysis to provide more in-depth knowledge of the mechanism. The work by Wu et al1 also adds novel concepts to therapeutic considerations. Taking into account the global concept that loss of function of IKs results in LQTS, while gain of function abnormalities cause short QT syndrome (SQTS), the authors consider the potential utility of drugs shown experimentally to act as IKs activators (to treat lossof-function mutations that cause LQTS) and suppressors (to treat gain-of-function mutations that cause SQTS). The idea, which is to some degree borne from the work on LQT3 by Schwartz and colleagues9 of the Long QT Registry. In that study, in which a gain-of-function sodium channel defect was treated with mexilitine, a sodium channel blocking agent, the treatment resulted in a shortening of the QT interval (to normal) and correction of the biophysical and electrocardiographic abnormalities.9 Using niflumic acid, an IKs activator, the authors showed that this drug can increase the current amplitude and shift the voltage dependence of activation in the negative direction in KCNQ1 and KCNQ1-KCNE1 wild-type channels and that the effects are amplified by the Y81C mutant. It is interesting to also note in this report that the IKs inhibitors HMR1556 and azimilide have differential effects on both wild-type channels and the Y81C mutant. In fact, the Y81C mutant reduced the effect of the IKs inhibitor HMR1556, while it enhanced the effect of the inhibitor azimilide. This observation supports the speculation that this mutation sensitizes the channel to drug response. Clearly, further work is needed to decipher the role of the functional nonsynonymous ion channel variants associated
Heart Rhythm, Vol 3, No 9, September 2006 with arrhythmogenesis for their ability to modulate the response to various antiarrhythmic drugs. This work suggests that drug effectiveness may rely on specific mutations, the biochemical (protein) alterations, and the position of the mutation within the channel. These studies may help to explain the biochemical and biophysical basis of subjective drug response and the paradoxical effects of drugs, which have profound implications for future management of patients including the potential for customized therapy.7
References 1.
2.
3. 4. 5.
6.
7.
8. 9.
Wu D-M, Lai L-P, Zhang M, Wang H-L, Jiang M, Liu X-S, Tseng G-N. Characterization of an LQT5-related mutation in KCNE1, Y81C: Implications for a role of KCNE1 cytoplasmic domain in IKs channel function. Heart Rhythm 2006;3:1031–1040. Lai LP, Su YN, Hsieh FJ, Chiang FT, Juang JM, Liu YB, Ho YL, Chen WJ, Yeh SJ, Wang CC, Ko YL, Wu TJ, Ueng KC, Lei MH, Tsao HM, Chen SA, Lin TK, Wu MH, Lo HM, Huang SK, Lin JL. Denaturing high-performance liquid chromatography screening of the long QT syndrome-related cardiac sodium and potassium channel genes and identification of novel mutations and single nucleotide polymorphisms. J Hum Genet 2005;50:490 – 496. Tapper AR, George AL Jr. MinK subdomains that mediate modulation of and association with KvLQT1. J Gen Physiol 2000;116:379 –390. Tapper AR, George AL Jr. Location and orientation of minK within the I(Ks) potassium channel complex. J Biol Chem 2001;276:38249 –38254. Melman YF, Krummerman A, McDonald TV. KCNE regulation of KvLQT1 channels: structure-function correlates. Trends Cardiovasc Med 2002;12:182– 187. Splawski I, Tristani-Firouzi M, Lehmann MH, Sanguinetti MC, Keating MT. Mutations in the hminK gene cause long QT syndrome and suppress IKs function. Nat Genet 1997;17:338 –340. Hong K, Piper DR, Diaz-Valdecantos A, Brugada J, Oliva A, Burashnikov E, Santos-de-Soto J, Grueso-Montero J, Diaz-Enfante E, Brugada P, Sachse F, Sanguinetti MC, Brugada R. De novo KCNQ1 mutation responsible for atrial fibrillation and short QT syndrome in utero. Cardiovasc Res 2005;68:433– 440. Delisle BP, Anson BD, Rajamani S, January CT. Biology of cardiac arrhythmias: ion channel protein trafficking. Circ Res 2004;94:1418 –1428. Schwartz PJ, Priori SG, Locati EH, Napolitano C, Cantu F, Towbin JA, Keating MT, Hammoude H, Brown AM, Chen LS. Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na⫹ channel blockade and to increases in heart rate. Implications for gene-specific therapy. Circulation 1995;92:3381–3386.