Mechanistic insights into robust cardiac IKs potassium channel activation by aromatic polyunsaturated fatty acid analogues

  1. Briana M Bohannon
  2. Jessica J Jowais
  3. Leif Nyberg
  4. Vanessa Olivier-Meo
  5. Valentina Corradi
  6. D Peter Tieleman
  7. Sara I Liin
  8. H Peter Larsson

  • Curated by eLife

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    eLife assessment

    This work reports important findings regarding the regulation of ion channels by polyunsaturated fatty acids (PUFAs) through the identification of novel aromatic PUFA analogs with potent effects on the IKs channels, which allow for mechanistic insights into their mode of action. The experiments are solid, combining site-directed mutagenesis, electrophysiological and pharmacological approaches to dissect the different molecular mechanisms and sites involved in the functional interactions. This work will be of broad interest to ion channel biophysicists, physiologists, and medical chemists interested in drug development for LQT syndrome. The study presents some limitations that may need to be addressed or further discussed, in order to strengthen the conclusions reached in the study.

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Abstract

Voltage-gated potassium (K V ) channels are important regulators of cellular excitability and control action potential repolarization in the heart and brain. K V channel mutations lead to disordered cellular excitability. Loss-of-function mutations, for example, result in membrane hyperexcitability, a characteristic of epilepsy and cardiac arrhythmias. Interventions intended to restore K V channel function have strong therapeutic potential in such disorders. Polyunsaturated fatty acids (PUFAs) and PUFA analogues comprise a class of K V channel activators with potential applications in the treatment of arrhythmogenic disorders such as long QT syndrome (LQTS). LQTS is caused by a loss-of-function of the cardiac I Ks channel – a tetrameric potassium channel complex formed by K V 7.1 and associated KCNE1 protein subunits. We have discovered a set of aromatic PUFA analogues that produce robust activation of the cardiac I Ks channel, and a unique feature of these PUFA analogues is an aromatic, tyrosine head group. We determine the mechanisms through which tyrosine PUFA analogues exert strong activating effects on the I Ks channel by generating modified aromatic head groups designed to probe cation–pi interactions, hydrogen bonding, and ionic interactions. We found that tyrosine PUFA analogues do not activate the I Ks channel through cation–pi interactions, but instead do so through a combination of hydrogen bonding and ionic interactions.

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  1. Version published to 10.7554/elife.85773 on eLife
  2. eLife

    eLife assessment

    This work reports important findings regarding the regulation of ion channels by polyunsaturated fatty acids (PUFAs) through the identification of novel aromatic PUFA analogs with potent effects on the IKs channels, which allow for mechanistic insights into their mode of action. The experiments are solid, combining site-directed mutagenesis, electrophysiological and pharmacological approaches to dissect the different molecular mechanisms and sites involved in the functional interactions. This work will be of broad interest to ion channel biophysicists, physiologists, and medical chemists interested in drug development for LQT syndrome. The study presents some limitations that may need to be addressed or further discussed, in order to strengthen the conclusions reached in the study.

  3. eLife

    Reviewer #1 (Public Review):

    This manuscript by Bohannon et al. continues a line of work from the Larsson laboratory with fundamental contributions describing the effects of polyunsaturated fatty acids (PUFAs) on the cardiac delayed rectifier potassium channel (IKs) formed by Kv7.1 and KCNE1 heteromers. Although the activating effect of PUFAs on these specific channels has been previously described, the authors now present a novel finding related to PUFAs containing large aromatic tyrosine head groups, showing significant activation effects on IKs, larger than other PUFAs previously studied. A combination of site-directed mutagenesis, electrophysiological and pharmacological approaches are used to dissect the different molecular mechanisms and sites involved in the functional interactions. The main conclusions are: 1) PUFA analogues with Tyr head groups are strong activators of the cardiac IKs channel by action on two previously described mechanisms: left-shift of the voltage-activation curve (by interaction with the voltage-sensor region of Kv7.1); and increased Gmax (by interacting with the pore region). 2) the underlying molecular interactions between PUFA and Kv7.1 are not cation-pi, as shown by the lack of effect of different chemical variations that disrupt the electrostatic surface potential. 3) the presence of electronegative groups on the aromatic ring favors increases in the maximal conductance. 4) the generation of a hydrogen bond with the -OH on the Tyr group seems to selectively impact on IKs voltage dependence of activation. 4) Kv7.1 sites involved in interactions with aromatic PUFAs are similar to the ones previously described for non-aromatic PUFAS, that is: R231 in S4 and K326 in S6. 5) residue T224 is newly identified as a potential site forming a hydrogen bond between the Tyr in the aromatic PUFA and Kv7.1.

    The manuscript is very well written and structured. The experiments are solid and lead to mostly well-grounded conclusions. There are some aspects that would benefit from some clarification, which are mainly related to the different effects of the aromatic PUFA variants on IKs voltage dependence and/or conductance.

  4. eLife

    Reviewer #2 (Public Review):

    In the present study, Briana M. Bohannon et al. expand on the study of the effect of Polyunsaturated fatty acids (PUFAS) on Iks (KV7.1 + KCNE1), a delayed rectifier potassium channel of critical relevance in cardiac physiology. PUFAs are amphipathic molecules that activate IKs channels by interacting with positively charged residues on the voltage sensor domain and in the channel's pore. The authors aim to characterize the molecular mechanisms behind the Iks activation by PUFA analogs that contains a tyrosine head group instead of the carboxyl or sulfonyl group present in other PUFAs.

    The authors present a well-written manuscript with clear data and well-presented figures. The authors describe the effects of various tyrosine-PUFA analogs and unveil the mechanistic nature of their interactions with the channel. The focus is the N -(alpha-linolenoyl) Tyrosine (NALT), a potent activator by shifting the channel G-V by more than 50mV facilitating the opening of the channel, although the authors tested other tyrosine-PUFA analogs. Remarkably, the hydroxyl group in the tyrosine head is essential to shift the voltage-dependence of activation due to an H-bond with a threonine from the S3-S4 linker that helps coordinate the PUFA together with an electrostatic interaction with arginine in the S4. Furthermore, to test whether the aromatic ring from the tyrosine had a role in the interaction, the authors took a fascinating and exciting approach by modifying it and making the ring more electronegative by adding negatively charged atoms. Interestingly, they discovered that an electronegative-modified aromatic PUFA could increase the channel's conductance, an effect mediated by a specific interaction with a Lysine at the top of the S6 helix.

    Although the question addressed in the manuscript is fascinating due to the possible use of these tyrosine-PUFA analogs as IKs modulators, the presented work is very mechanistic and specialized. While the effect of tyrosine-PUFA analogs is robust, the authors could improve the story by highlighting their interest in them and discussing whether they have potential therapeutic uses.

    Due to the relevance of IKs currents in cardiac physiology and Long QT syndrome, the discovery and characterization of activators are highly relevant. The present manuscript presents a group of potent IKs channel activators that have the potential to impact the cardiac physiology field dramatically if they can perform under pathophysiological conditions or in the presence of disease-causing mutations.

  5. eLife

    Reviewer #3 (Public Review):

    Bohannon and colleagues demonstrate that aromatic PUFA analogues positively modulate delayed rectifier potassium channel (Iks) currents, identifying new compounds that could be useful for the treatment of long QT syndrome. The data suggest that aromatic PUFA analogues have two modulatory effects that occur by distinct mechanisms involving hydrogen bonds and ionic interactions. However, the exact determinants of these molecular interactions remain unclear.

    Strengths of the study include the following:

    1. By examining a large panel of aromatic PUFA analogues, the study provides a thorough understanding of the relationship between the structure of these analogues and the modulatory effect. Of note, these aromatic PUFA analogues are more efficacious than previously characterized PUFAs such as DHA and N-AT. This knowledge will be important for the design of PUFA analogues for the modulation of IKs current, which could be a strategy for the treatment of long QT syndrome.
    2. By examining the effect of mutations previously shown to disrupt two mechanisms of PUFA modulation, the results suggest that aromatic PUFAs act through the same mechanisms. Furthermore, the effects of the different analogues shed light on the determinants of these binding sites such as the presence of additional hydrogen bonds and electrostatic interactions between the aromatic PUFAs and ion channels.

    One limitation of the study is that the structure-activity relationships and effects of the mutations do not provide a complete molecular understanding of how the aromatic PUFA analogues are interacting with the channel. This understanding will require additional studies to examine PUFA analogue binding combined with more extensive mutagenesis. Specifically, the model in Figure 5 suggests that the effect of aromatic PUFAs on the voltage dependence of activation depends on an electrostatic interaction with R231 and a hydrogen bond interaction possibly with T224. Similarly, the effect on channel conductance depends on an electrostatic interaction with K326 through the carboxylate anion of the aromatic PUFA as well as an additional electrostatic interaction with some other part of the protein. It is unclear what residues mediate these interactions. Additionally, the authors propose that T224 is forming a hydrogen bond interaction with the hydroxyl group of NALT, but there appears to be a relatively similar effect of the T224V mutation on NAL-phe, only that the spread in the data makes this effect statistically insignificant. Therefore, the conclusion that T224 mediates NALT action by forming a hydrogen bond with the hydroxyl group (a chemical moiety that is absent in NAL-phe) is not fully supported by the data. A structural model to indicate that T224 is well-positioned to form a hydrogen bond with NALT when it is also interacting with R231 would strengthen this model.

  6. Version published to 10.1101/2023.01.12.523777v1 on bioRxiv