- Email: [email protected]
Say Cheese: Structure of the Cardiac Electrical Engine Is Captured
TIBS 1652 No. of Pages 3
Trends in Biochemical Sciences An official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECUL AR BIOLOGY
Spotlight
Say Cheese: Structure of the Cardiac Electrical Engine Is Captured Mohammad-Reza Ghovanloo and Peter C. Ruben1,*
1
Voltage-gated sodium channel (Nav)1.5 is the predominantly expressed sodium channel in the myocardium. Mutations in the gene encoding Nav1.5 are associated with several types of cardiac arrhythmias. In their recent study, Jiang et al. provide a detailed structure of the rat Nav1.5, with major implications regarding its physiology, pharmacology, and pathophysiology. Electrical signalling is a vital part of human physiology. The nature of electrical signalling remained a mystery until the masterful work of Hodgkin and Huxley from 1949 to 1952 that described sodium and potassium conductances regulated by a series of particles [1]. For those interested in the sodium conductance, the m and h particles, which we now call Navs, were particularly interesting. Today, in addition to making inroads into Nav function, we can also visualise their structures at the atomic level, thanks in large part to the efforts spearheaded by Catterall and colleagues in the early 2010s [2,3]. Gaining structural insight into Navs has been a long journey, in part due to their large size. In 2011, Payandeh et al. solved the first crystal structure of Arcobacter butzleri Nav (NavAb) [2]. The NavAb structure revealed abundant information about structure–function relationships in Navs; however, the prokaryotic homotetramer NavAb lacks several key features of eukaryotic Navs, including fast inactivation and charge and size asymmetry in voltage sensors. Recently, multiple cryo-electron
mediate fast inactivation. Jiang et al. generated a model of fast inactivation using the activated Nav1.5 structure and the previously published resting NavAb structure. Their results further suggest that the outward movement of the S4 segment of the third domain triggers opening of an allosteric binding site for the IFM motif of the fast inactivation particle. Of course, this interaction also requires the outward movement of the voltage sensor of the fourth In their recent study, Jiang et al. deter- domain to shift the position of the fast mined the structure of the rat Nav1.5 at inactivation particle (last event in the fast 3.2–3.5 Å resolution [9]. They made sev- inactivation pathway). These findings eral comparisons to previously published are consistent with previously proposed structures, including human Nav1.7 mechanisms for fast inactivation [4–8]. (found in the peripheral nervous system) and the resting-state structure of NavAb To be considered a Nav, a channel must [9,10]. This approach elucidated the struc- preferentially conduct sodium. This crucial tural basis for several key properties of property is a direct result of the sequence Nav1.5, as well as implications on the and structure of the Nav selectivity filter. Nav1.5 physiology, pharmacology, and The mechanisms of sodium selectivity and pathophysiology. conductance have long been important topics of research. The ‘dunked’ conformaThere are four main Nav β subtypes; β1/3 tion of a lysine residue on the innermost seginteract noncovalently, and β2/4 interact ment of the Nav1.5 selectivity filter suggests covalently with the α subunit. Earlier func- an important role in the course of the sodium tional studies showed that β subunits do permeation pathway. Jiang et al. propose a not significantly change either kinetic or mechanism in which the side chain of this lyvoltage-dependent properties of Nav1.5 sine, located deep inside the selectivity filter, [10]. Importantly, Jiang et al. determined forms a complex where the positive charge the structural basis for reduced modulation (NH3+) is delocalized by interactions with of Nav1.5 by β subunits; an asparagine backbone carbonyl groups of nearby resi(N-linked glycosylated) in the β1/3 inter- dues. This delocalization in turn allows the action site may disrupt the noncovalent free electrons on the nitrogen of lysine to cointeraction between the α and β1/3 sub- ordinate the movement of sodium ions. units. Likewise, the replacement of a key cysteine with leucine in Nav1.5 destabi- Nav1.5 also has a unique relationship with lizes covalent interactions between the α tetrodotoxin (TTX). Among the mammalian and β2/4 subunits. Nav, only Nav1.5, Nav1.8, and Nav1.9 (PNS subtypes) are considered TTX resisThe conformational changes in the four tant (TTX-R) [1]. The molecular reason unpore domains of Navs are linked to the po- derlying differential affinity for TTX in Navs sition and configurations of respective has been attributed predominantly to a voltage-sensing domains. The sliding- single homologous residue difference in helix model of Nav gating suggests that, the Nav pore loop. The TTX-R channels upon depolarization, an outward move- have a cysteine or serine in this position, ment of the voltage sensors pulls on S4– instead of a tyrosine or phenylalanine resiS5 linkers, which in turn open the pore do- due in TTX-sensitive channels. Jiang et al. mains. This activity at domains III and IV determined that this substitution does not microscopy (cryo-EM) structures of eukaryotic Navs have been published, including the skeletal muscle and neuronal subtypes [4–8]. However, since the flurry of cryo-EM structures began, cardiac electrophysiologists have been eagerly awaiting the Nav1.5 structure. Nav1.5 has several unique properties relative to other Navs, including its relationship with its β subunits.
Trends in Biochemical Sciences, Month 2020, Vol. xx, No. xx
1
Trends in Biochemical Sciences An official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECUL AR BIOLOGY
interaction with an innermost lysine residue on the selectivity filter reveals flecainide’s mechanism of sodium conductance inhibition through Nav1.5. The structural insights into both pore-domain receptor sites (outer selectivity filter TTX is considered a state-independent and local-anaesthetic receptor site) has Nav blocker, a function of its binding site important pharmacological implications residing on the outer selectivity filter, for drug design. which is a more rigid part of the Nav pore domain. By contrast, most local anaes- Malignant mutations in SCN5A, the gene thetics and antiarrhythmics are highly that encodes Nav1.5, alter phase 0 of the state-dependent Nav blockers; their cardiac action potential. These mutations binding site is located below the selectivity alter Nav excitability and can cause filter, a more flexible region of the pore cardiac arrhythmias (e.g., long QT-3 domain. Jiang et al. provided a high- syndrome), some of which may be fatal. resolution view of this antiarrhythmic/ Jiang et al. also studied the effects of sevlocal-anaesthetic receptor site in Nav1.5 eral arrhythmic mutations on the atomic bound with flecainide; its piperidine structure of Nav1.5, including possible alter the local conformation of the channel. However, lacking an aromatic side chain in this position may cause steric constraints that reduce the TTX affinity in Nav1.5.
roles of pathogenic gating-pore currents in arrhythmia, providing important clinical insights. It is worth noting that rat and human Nav1.5s are 94% homologous, so what we have learned from Jiang et al. has clinical relevance. The findings in the recent publication by Jiang et al. are plentiful, with a wide range of implications ranging from Nav1.5 physiology to pharmacology and pathophysiology. However, some mysteries still abound. The structures of extracellular and intracellular linkers, the latter of which contain key structures, including the calmodulin binding sites, have yet to be resolved. However, the paper by Jiang et al. provides important tools with which ion channel biophysicists,
Trends in Biochemical Sciences
Figure 1. Key Findings from Structural Study of Nav1.5. This is a cartoon summarizing key direct findings of the recent study by Jiang et al., [9] and the major implications of the study broadly relating to the fields of voltage-gated sodium channel (Nav) physiology, pharmacology, and pathophysiology. In the middle is the cryo-electron microscopic structure of Nav1.5 (PDB ID: 6UZ3), with domain (D) I in orange, DII in cyan, DIII in blue, and DIV in pink. Also marked are the voltage-sensing domain (VSD), the pore domain (PD), and the sodium channel. On the right there are sample macroscopic sodium currents from wild type, a long QT-3 (LQT-3) causing mutation, and effects of flecainide on the channel, as would be obtained from voltage-clamp experiments. To the right of the sodium current traces are corresponding ventricular action potential traces, as would be obtained from current-clamp experiments. These traces are meant to show sample manifestations of mutational [e.g., LQT-3 mutant increases persistent currents, which may prolong the action potential (AP) duration] and pharmacological (e.g., flecainide blocks peak and persistent currents, which may restore AP duration to wild type) modulation of Nav1.5, with the findings from Jiang et al. serving as tools for better understanding these manifestations.
2
Trends in Biochemical Sciences, Month 2020, Vol. xx, No. xx
Trends in Biochemical Sciences An official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECUL AR BIOLOGY
cardiac researchers, pharmacologists, and clinicians will find answers to many long-standing questions (Figure 1).
*Correspondence: [email protected] (P.C. Ruben). https://doi.org/10.1016/j.tibs.2020.02.003 © 2020 Elsevier Ltd. All rights reserved.
Acknowledgements This work was supported by grants from the Natural Science and Engineering Research Council of Canada and the Rare Disease Foundation to P.C.R. and M-R.G. (CGS-D & MSFSS).
1
Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada
Pan, X. et al. (2018) Structure of the human voltage-gated sodium channel Nav1.4 in complex with β1. Science 362, eaau2486 6. Yan, Z. et al. (2017) Structure of the Nav1.4-β1 complex from electric eel. Cell 170, 470–482.e11 7. Pan, X. et al. (2019) Molecular basis for pore blockade of human Na + channel Na v 1.2 by the m-conotoxin KIIIA. Science 363, 1309–1313 8. Shen, H. et al. (2019) Structures of human Na v 1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303–1308 9. Jiang, D. et al. (2020) Structure of the cardiac sodium channel. Cell 180, 122–134 10. Wisedchaisri, G. et al. (2019) Resting-state structure and gating mechanism of a voltage-gated sodium channel. Cell 178, 993–1003.e12
5.
References 1. Hille, B. (2001) Ion Channels of Excitable Membranes, Sinauer 2. Payandeh, J. et al. (2011) The crystal structure of a voltage-gated sodium channel. Nature 475, 353–359 3. Catterall, W.A. (2014) Structure and function of voltage-gated sodium channels at atomic resolution. Exp. Physiol. 99, 35–51 4. Shen, H. et al. (2017) Structure of a eukaryotic voltagegated sodium channel at near atomic resolution. Science 355, 1–19
Trends in Biochemical Sciences, Month 2020, Vol. xx, No. xx
3
Trends in Biochemical Sciences An official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECUL AR BIOLOGY
Spotlight
Say Cheese: Structure of the Cardiac Electrical Engine Is Captured Mohammad-Reza Ghovanloo and Peter C. Ruben1,*
1
Voltage-gated sodium channel (Nav)1.5 is the predominantly expressed sodium channel in the myocardium. Mutations in the gene encoding Nav1.5 are associated with several types of cardiac arrhythmias. In their recent study, Jiang et al. provide a detailed structure of the rat Nav1.5, with major implications regarding its physiology, pharmacology, and pathophysiology. Electrical signalling is a vital part of human physiology. The nature of electrical signalling remained a mystery until the masterful work of Hodgkin and Huxley from 1949 to 1952 that described sodium and potassium conductances regulated by a series of particles [1]. For those interested in the sodium conductance, the m and h particles, which we now call Navs, were particularly interesting. Today, in addition to making inroads into Nav function, we can also visualise their structures at the atomic level, thanks in large part to the efforts spearheaded by Catterall and colleagues in the early 2010s [2,3]. Gaining structural insight into Navs has been a long journey, in part due to their large size. In 2011, Payandeh et al. solved the first crystal structure of Arcobacter butzleri Nav (NavAb) [2]. The NavAb structure revealed abundant information about structure–function relationships in Navs; however, the prokaryotic homotetramer NavAb lacks several key features of eukaryotic Navs, including fast inactivation and charge and size asymmetry in voltage sensors. Recently, multiple cryo-electron
mediate fast inactivation. Jiang et al. generated a model of fast inactivation using the activated Nav1.5 structure and the previously published resting NavAb structure. Their results further suggest that the outward movement of the S4 segment of the third domain triggers opening of an allosteric binding site for the IFM motif of the fast inactivation particle. Of course, this interaction also requires the outward movement of the voltage sensor of the fourth In their recent study, Jiang et al. deter- domain to shift the position of the fast mined the structure of the rat Nav1.5 at inactivation particle (last event in the fast 3.2–3.5 Å resolution [9]. They made sev- inactivation pathway). These findings eral comparisons to previously published are consistent with previously proposed structures, including human Nav1.7 mechanisms for fast inactivation [4–8]. (found in the peripheral nervous system) and the resting-state structure of NavAb To be considered a Nav, a channel must [9,10]. This approach elucidated the struc- preferentially conduct sodium. This crucial tural basis for several key properties of property is a direct result of the sequence Nav1.5, as well as implications on the and structure of the Nav selectivity filter. Nav1.5 physiology, pharmacology, and The mechanisms of sodium selectivity and pathophysiology. conductance have long been important topics of research. The ‘dunked’ conformaThere are four main Nav β subtypes; β1/3 tion of a lysine residue on the innermost seginteract noncovalently, and β2/4 interact ment of the Nav1.5 selectivity filter suggests covalently with the α subunit. Earlier func- an important role in the course of the sodium tional studies showed that β subunits do permeation pathway. Jiang et al. propose a not significantly change either kinetic or mechanism in which the side chain of this lyvoltage-dependent properties of Nav1.5 sine, located deep inside the selectivity filter, [10]. Importantly, Jiang et al. determined forms a complex where the positive charge the structural basis for reduced modulation (NH3+) is delocalized by interactions with of Nav1.5 by β subunits; an asparagine backbone carbonyl groups of nearby resi(N-linked glycosylated) in the β1/3 inter- dues. This delocalization in turn allows the action site may disrupt the noncovalent free electrons on the nitrogen of lysine to cointeraction between the α and β1/3 sub- ordinate the movement of sodium ions. units. Likewise, the replacement of a key cysteine with leucine in Nav1.5 destabi- Nav1.5 also has a unique relationship with lizes covalent interactions between the α tetrodotoxin (TTX). Among the mammalian and β2/4 subunits. Nav, only Nav1.5, Nav1.8, and Nav1.9 (PNS subtypes) are considered TTX resisThe conformational changes in the four tant (TTX-R) [1]. The molecular reason unpore domains of Navs are linked to the po- derlying differential affinity for TTX in Navs sition and configurations of respective has been attributed predominantly to a voltage-sensing domains. The sliding- single homologous residue difference in helix model of Nav gating suggests that, the Nav pore loop. The TTX-R channels upon depolarization, an outward move- have a cysteine or serine in this position, ment of the voltage sensors pulls on S4– instead of a tyrosine or phenylalanine resiS5 linkers, which in turn open the pore do- due in TTX-sensitive channels. Jiang et al. mains. This activity at domains III and IV determined that this substitution does not microscopy (cryo-EM) structures of eukaryotic Navs have been published, including the skeletal muscle and neuronal subtypes [4–8]. However, since the flurry of cryo-EM structures began, cardiac electrophysiologists have been eagerly awaiting the Nav1.5 structure. Nav1.5 has several unique properties relative to other Navs, including its relationship with its β subunits.
Trends in Biochemical Sciences, Month 2020, Vol. xx, No. xx
1
Trends in Biochemical Sciences An official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECUL AR BIOLOGY
interaction with an innermost lysine residue on the selectivity filter reveals flecainide’s mechanism of sodium conductance inhibition through Nav1.5. The structural insights into both pore-domain receptor sites (outer selectivity filter TTX is considered a state-independent and local-anaesthetic receptor site) has Nav blocker, a function of its binding site important pharmacological implications residing on the outer selectivity filter, for drug design. which is a more rigid part of the Nav pore domain. By contrast, most local anaes- Malignant mutations in SCN5A, the gene thetics and antiarrhythmics are highly that encodes Nav1.5, alter phase 0 of the state-dependent Nav blockers; their cardiac action potential. These mutations binding site is located below the selectivity alter Nav excitability and can cause filter, a more flexible region of the pore cardiac arrhythmias (e.g., long QT-3 domain. Jiang et al. provided a high- syndrome), some of which may be fatal. resolution view of this antiarrhythmic/ Jiang et al. also studied the effects of sevlocal-anaesthetic receptor site in Nav1.5 eral arrhythmic mutations on the atomic bound with flecainide; its piperidine structure of Nav1.5, including possible alter the local conformation of the channel. However, lacking an aromatic side chain in this position may cause steric constraints that reduce the TTX affinity in Nav1.5.
roles of pathogenic gating-pore currents in arrhythmia, providing important clinical insights. It is worth noting that rat and human Nav1.5s are 94% homologous, so what we have learned from Jiang et al. has clinical relevance. The findings in the recent publication by Jiang et al. are plentiful, with a wide range of implications ranging from Nav1.5 physiology to pharmacology and pathophysiology. However, some mysteries still abound. The structures of extracellular and intracellular linkers, the latter of which contain key structures, including the calmodulin binding sites, have yet to be resolved. However, the paper by Jiang et al. provides important tools with which ion channel biophysicists,
Trends in Biochemical Sciences
Figure 1. Key Findings from Structural Study of Nav1.5. This is a cartoon summarizing key direct findings of the recent study by Jiang et al., [9] and the major implications of the study broadly relating to the fields of voltage-gated sodium channel (Nav) physiology, pharmacology, and pathophysiology. In the middle is the cryo-electron microscopic structure of Nav1.5 (PDB ID: 6UZ3), with domain (D) I in orange, DII in cyan, DIII in blue, and DIV in pink. Also marked are the voltage-sensing domain (VSD), the pore domain (PD), and the sodium channel. On the right there are sample macroscopic sodium currents from wild type, a long QT-3 (LQT-3) causing mutation, and effects of flecainide on the channel, as would be obtained from voltage-clamp experiments. To the right of the sodium current traces are corresponding ventricular action potential traces, as would be obtained from current-clamp experiments. These traces are meant to show sample manifestations of mutational [e.g., LQT-3 mutant increases persistent currents, which may prolong the action potential (AP) duration] and pharmacological (e.g., flecainide blocks peak and persistent currents, which may restore AP duration to wild type) modulation of Nav1.5, with the findings from Jiang et al. serving as tools for better understanding these manifestations.
2
Trends in Biochemical Sciences, Month 2020, Vol. xx, No. xx
Trends in Biochemical Sciences An official publication of the INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECUL AR BIOLOGY
cardiac researchers, pharmacologists, and clinicians will find answers to many long-standing questions (Figure 1).
*Correspondence: [email protected] (P.C. Ruben). https://doi.org/10.1016/j.tibs.2020.02.003 © 2020 Elsevier Ltd. All rights reserved.
Acknowledgements This work was supported by grants from the Natural Science and Engineering Research Council of Canada and the Rare Disease Foundation to P.C.R. and M-R.G. (CGS-D & MSFSS).
1
Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada
Pan, X. et al. (2018) Structure of the human voltage-gated sodium channel Nav1.4 in complex with β1. Science 362, eaau2486 6. Yan, Z. et al. (2017) Structure of the Nav1.4-β1 complex from electric eel. Cell 170, 470–482.e11 7. Pan, X. et al. (2019) Molecular basis for pore blockade of human Na + channel Na v 1.2 by the m-conotoxin KIIIA. Science 363, 1309–1313 8. Shen, H. et al. (2019) Structures of human Na v 1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303–1308 9. Jiang, D. et al. (2020) Structure of the cardiac sodium channel. Cell 180, 122–134 10. Wisedchaisri, G. et al. (2019) Resting-state structure and gating mechanism of a voltage-gated sodium channel. Cell 178, 993–1003.e12
5.
References 1. Hille, B. (2001) Ion Channels of Excitable Membranes, Sinauer 2. Payandeh, J. et al. (2011) The crystal structure of a voltage-gated sodium channel. Nature 475, 353–359 3. Catterall, W.A. (2014) Structure and function of voltage-gated sodium channels at atomic resolution. Exp. Physiol. 99, 35–51 4. Shen, H. et al. (2017) Structure of a eukaryotic voltagegated sodium channel at near atomic resolution. Science 355, 1–19
Trends in Biochemical Sciences, Month 2020, Vol. xx, No. xx
3