Publications by authors named "Harley T Kurata"

58 Publications

Unconventional voltage sensing in an inwardly rectifying potassium channel.

Authors:
Harley T Kurata

J Gen Physiol 2021 Jun 6;153(6). Epub 2021 May 6.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

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http://dx.doi.org/10.1085/jgp.202112929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105720PMC
June 2021

Familial neonatal seizures caused by the Kv7.3 selectivity filter mutation T313I.

Epilepsia Open 2020 Dec 17;5(4):562-573. Epub 2020 Oct 17.

Department of Pharmacology Alberta Diabetes Institute University of Alberta Edmonton AB Canada.

Objective: A spectrum of seizure disorders is linked to mutations in Kv7.2 and Kv7.3 channels. Linking functional effects of identified mutations to their clinical presentation requires ongoing characterization of newly identified variants. In this study, we identified and functionally characterized a previously unreported mutation in the selectivity filter of Kv7.3.

Methods: Next-generation sequencing was used to identify the Kv7.3[T313I] mutation in a family affected by neonatal seizures. Electrophysiological approaches were used to characterize the functional effects of this mutation on ion channels expressed in oocytes.

Results: Substitution of residue 313 from threonine to isoleucine (Kv7.3[T313I]) likely disrupts a critical intersubunit hydrogen bond. Characterization of the mutation in homomeric Kv7.3 channels demonstrated a total loss of channel function. Assembly in heteromeric channels (with Kv7.2) leads to modest suppression of total current when expressed in oocytes. Using a Kv7 activator with distinct effects on homomeric Kv7.2 vs heteromeric Kv7.2/Kv7.3 channels, we demonstrated that assembly of Kv7.2 and Kv7.3[T313I] generates functional channels.

Significance: Biophysical and clinical effects of the T313I mutation are consistent with Kv7.3 mutations previously identified in cases of pharmacoresponsive self-limiting neonatal epilepsy. These findings expand our description of functionally characterized Kv7 channel variants and report new methods to distinguish molecular mechanisms of channel mutations.
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http://dx.doi.org/10.1002/epi4.12438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733659PMC
December 2020

Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels.

Elife 2020 11 9;9. Epub 2020 Nov 9.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Canada.

Many voltage-dependent ion channels are regulated by accessory proteins. We recently reported powerful regulation of Kv1.2 potassium channels by the amino acid transporter Slc7a5. In this study, we report that Kv1.1 channels are also regulated by Slc7a5, albeit with different functional outcomes. In heterologous expression systems, Kv1.1 exhibits prominent current enhancement ('disinhibition') with holding potentials more negative than -120 mV. Knockdown of endogenous Slc7a5 leads to larger Kv1.1 currents and strongly attenuates the disinhibition effect, suggesting that Slc7a5 regulation of Kv1.1 involves channel inhibition that can be reversed by supraphysiological hyperpolarizing voltages. We investigated chimeric combinations of Kv1.1 and Kv1.2, demonstrating that exchange of the voltage-sensing domain controls the sensitivity and response to Slc7a5, and localize a specific position in S1 with prominent effects on Slc7a5 sensitivity. Overall, our study highlights multiple Slc7a5-sensitive Kv1 subunits, and identifies the voltage-sensing domain as a determinant of Slc7a5 modulation of Kv1 channels.
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http://dx.doi.org/10.7554/eLife.54916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7690953PMC
November 2020

A structure-based computational workflow to predict liability and binding modes of small molecules to hERG.

Sci Rep 2020 10 1;10(1):16262. Epub 2020 Oct 1.

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada.

Off-target interactions of drugs with the human ether-à-go-go related gene 1 (hERG1) channel have been associated with severe cardiotoxic conditions leading to the withdrawal of many drugs from the market over the last decades. Consequently, predicting drug-induced hERG-liability is now a prerequisite in any drug discovery campaign. Understanding the atomic level interactions of drug with the channel is essential to guide the efficient development of safe drugs. Here we utilize the recent cryo-EM structure of the hERG channel and describe an integrated computational workflow to characterize different drug-hERG interactions. The workflow employs various structure-based approaches and provides qualitative and quantitative insights into drug binding to hERG. Our protocol accurately differentiated the strong blockers from weak and revealed three potential anchoring sites in hERG. Drugs engaging in all these sites tend to have high affinity towards hERG. Our results were cross-validated using a fluorescence polarization kit binding assay and with electrophysiology measurements on the wild-type (WT-hERG) and on the two hERG mutants (Y652A-hERG and F656A-hERG), using the patch clamp technique on HEK293 cells. Finally, our analyses show that drugs binding to hERG disrupt and hijack certain native-structural networks in the channel, thereby, gaining more affinity towards hERG.
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http://dx.doi.org/10.1038/s41598-020-72889-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530726PMC
October 2020

Endoplasmic reticulum stress in the dorsal root ganglia regulates large-conductance potassium channels and contributes to pain in a model of multiple sclerosis.

FASEB J 2020 09 17;34(9):12577-12598. Epub 2020 Jul 17.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, AB, Canada.

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca -sensitive BK-type K channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.
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http://dx.doi.org/10.1096/fj.202001163RDOI Listing
September 2020

Functional and behavioral signatures of Kv7 activator drug subtypes.

Epilepsia 2020 08 11;61(8):1678-1690. Epub 2020 Jul 11.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.

Objective: Voltage-gated potassium channels of the KCNQ (Kv7) family are targeted by a variety of activator compounds with therapeutic potential for treatment of epilepsy. Exploration of this drug class has revealed a variety of effective compounds with diverse mechanisms. In this study, we aimed to clarify functional criteria for categorization of Kv7 activator compounds, and to compare the effects of prototypical drugs in a zebrafish larvae model.

Methods: In vitro electrophysiological approaches with recombinant ion channels were used to highlight functional properties important for classification of drug mechanisms. We also benchmarked the effects of representative antiepileptic Kv7 activator drugs using behavioral seizure assays of zebrafish larvae and in vivo Ca imaging with the ratiometric Ca sensor CaMPARI.

Results: Drug effects on channel gating kinetics, and drug sensitivity profiles to diagnostic channel mutations, were used to highlight properties for categorization of Kv7 activator drugs into voltage sensor-targeted or pore-targeted subtypes. Quantifying seizures and ratiometric Ca imaging in freely swimming zebrafish larvae demonstrated that while all Kv7 activators tested lead to suppression of neuronal excitability, pore-targeted activators (like ML213 and retigabine) strongly suppress seizure behavior, whereas ICA-069673 triggers a seizure-like hypermotile behavior.

Significance: This study suggests criteria to categorize antiepileptic Kv7 activator drugs based on their underlying mechanism. We also establish the use of in vivo CaMPARI as a tool for screening effects of anticonvulsant drugs on neuronal excitability in zebrafish. In summary, despite a shared ability to suppress neuronal excitability, our findings illustrate how mechanistic differences between Kv7 activator subtypes influence their effects on heteromeric channels and lead to vastly different in vivo outcomes.
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http://dx.doi.org/10.1111/epi.16592DOI Listing
August 2020

Heteromeric Assembly of Truncated Neuronal Kv7 Channels: Implications for Neurologic Disease and Pharmacotherapy.

Mol Pharmacol 2020 09 24;98(3):192-202. Epub 2020 Jun 24.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada (J.L., J.M., S.M.L., H.T.K.) and Department of Neurodevelopmental Medicine, Cortica Healthcare, San Rafael, California (E.J.M.)

Neuronal voltage-gated potassium channels (Kv) are critical regulators of electrical activity in the central nervous system. Mutations in the KCNQ (Kv7) ion channel family are linked to epilepsy and neurodevelopmental disorders. These channels underlie the neuronal "M-current" and cluster in the axon initial segment to regulate the firing of action potentials. There is general consensus that KCNQ channel assembly and heteromerization are controlled by C-terminal helices. We identified a pediatric patient with neurodevelopmental disability, including autism traits, inattention and hyperactivity, and ataxia, who carries a de novo frameshift mutation in KCNQ3 (KCNQ3-FS534), leading to truncation of ∼300 amino acids in the C terminus. We investigated possible molecular mechanisms of channel dysfunction, including haplo-insufficiency or a dominant-negative effect caused by the assembly of truncated KCNQ3 and functional KCNQ2 subunits. We also used a recently recognized property of the KCNQ2-specific activator ICA-069673 to identify assembly of heteromeric channels. ICA-069673 exhibits a functional signature that depends on the subunit composition of KCNQ2/3 channels, allowing us to determine whether truncated KCNQ3 subunits can assemble with KCNQ2. Our findings demonstrate that although the KCNQ3-FS534 mutant does not generate functional channels on its own, large C-terminal truncations of KCNQ3 (including the KCNQ3-FS534 mutation) assemble efficiently with KCNQ2 but fail to promote or stabilize KCNQ2/KCNQ3 heteromeric channel expression. Therefore, the frequent assumption that pathologies linked to KCNQ3 truncations arise from haplo-insufficiency should be reconsidered in some cases. Subtype-specific channel activators like ICA-069673 are a reliable tool to identify heteromeric assembly of KCNQ2 and KCNQ3. SIGNIFICANCE STATEMENT: Mutations that truncate the C terminus of neuronal Kv7/KCNQ channels are linked to a spectrum of seizure disorders. One role of the multifunctional KCNQ C terminus is to mediate subtype-specific assembly of heteromeric KCNQ channels. This study describes the use of a subtype-specific Kv7 activator to assess assembly of heteromeric KCNQ2/KCNQ3 (Kv7.2/Kv7.3) channels and demonstrates that large disease-linked and experimentally generated C-terminal truncated KCNQ3 mutants retain the ability to assemble with KCNQ2.
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http://dx.doi.org/10.1124/mol.120.119644DOI Listing
September 2020

Chemical regulation of Kv7 channels: Diverse scaffolds, sites, and mechanisms of action.

Authors:
Harley T Kurata

J Gen Physiol 2020 08;152(8)

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

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http://dx.doi.org/10.1085/jgp.202012598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7398145PMC
August 2020

Slc7a5 alters Kvβ-mediated regulation of Kv1.2.

J Gen Physiol 2020 07;152(7)

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.

The voltage-gated potassium channel Kv1.2 plays a pivotal role in neuronal excitability and is regulated by a variety of known and unknown extrinsic factors. The canonical accessory subunit of Kv1.2, Kvβ, promotes N-type inactivation and cell surface expression of the channel. We recently reported that a neutral amino acid transporter, Slc7a5, alters the function and expression of Kv1.2. In the current study, we investigated the effects of Slc7a5 on Kv1.2 in the presence of Kvβ1.2 subunits. We observed that Slc7a5-induced suppression of Kv1.2 current and protein expression was attenuated with cotransfection of Kvβ1.2. However, gating effects mediated by Slc7a5, including disinhibition and a hyperpolarizing shift in channel activation, were observed together with Kvβ-mediated inactivation, indicating convergent regulation of Kv1.2 by both regulatory proteins. Slc7a5 influenced several properties of Kvβ-induced inactivation of Kv1.2, including accelerated inactivation, a hyperpolarizing shift and greater extent of steady-state inactivation, and delayed recovery from inactivation. These modified inactivation properties were also apparent in altered deactivation of the Kv1.2/Kvβ/Slc7a5 channel complex. Taken together, these findings illustrate a functional interaction arising from simultaneous regulation of Kv1.2 by Kvβ and Slc7a5, leading to powerful effects on Kv1.2 expression, gating, and overall channel function.
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http://dx.doi.org/10.1085/jgp.201912524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335012PMC
July 2020

Sensory Neurons of the Dorsal Root Ganglia Become Hyperexcitable in a T-Cell-Mediated MOG-EAE Model of Multiple Sclerosis.

eNeuro 2019 Mar-Apr;6(2). Epub 2019 Apr 1.

Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Alberta T6G 2E1, Canada.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system. Patients with MS typically present with visual, motor, and sensory deficits. However, an additional complication of MS in large subset of patients is neuropathic pain. To study the underlying immune-mediated pathophysiology of pain in MS we employed the myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE) model in mice. Since sensory neurons are crucial for nociceptive transduction, we investigated the effect of this disease on sensory neurons of the lumbar dorsal root ganglia (DRG). Here, we report the disease was associated with activation of the complement system and the NLRP3 inflammasome in the DRG. We further observe a transient increase in the number of complement component 5a receptor 1-positive (C5aR1+) immune cells, CD4+ T-cells, and Iba1+ macrophages in the DRG. The absence of any significant change in the levels of mRNA for myelin proteins in the DRG and the sciatic nerve suggests that demyelination in the PNS is not a trigger for the immune response in the DRG. However, we did observe an induction of activating transcription factor 3 (ATF3) at disease onset and chronic disruption of cytoskeletal proteins in the DRG demonstrating neuronal injury in the PNS in response to the disease. Electrophysiological analysis revealed the emergence of hyperexcitability in medium-to-large (≥26 µm) diameter neurons, especially at the onset of MOG-EAE signs. These results provide conclusive evidence of immune activation, neuronal injury, and peripheral sensitization in MOG-EAE, a model classically considered to be centrally mediated.
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http://dx.doi.org/10.1523/ENEURO.0024-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6449162PMC
March 2020

Pore- and voltage sensor-targeted KCNQ openers have distinct state-dependent actions.

J Gen Physiol 2018 12 29;150(12):1722-1734. Epub 2018 Oct 29.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada

Ion channels encoded by generate a prominent K conductance in the central nervous system, referred to as the M current, which is controlled by membrane voltage and PIP2. The KCNQ2-5 voltage-gated potassium channels are targeted by a variety of activating compounds that cause negative shifts in the voltage dependence of activation. The underlying pharmacology of these effects is of growing interest because of possible clinical applications. Recent studies have revealed multiple binding sites and mechanisms of action of KCNQ activators. For example, retigabine targets the pore domain, but several compounds have been shown to influence the voltage-sensing domain. An important unexplored feature of these compounds is the influence of channel gating on drug binding or effects. In the present study, we compare the state-dependent actions of retigabine and ICA-069673 (ICA73, a voltage sensor-targeted activator). We assess drug binding to preopen states by applying drugs to homomeric KCNQ2 channels at different holding voltages, demonstrating little or no association of ICA73 with resting states. Using rapid solution switching, we also demonstrate that the rate of onset of ICA73 correlates with the voltage dependence of channel activation. Retigabine actions differ significantly, with prominent drug effects seen at very negative holding voltages and distinct voltage dependences of drug binding versus channel activation. Using similar approaches, we investigate the mechanistic basis for attenuation of ICA73 actions by the voltage-sensing domain mutation KCNQ2[A181P]. Our findings demonstrate different state-dependent actions of pore- versus voltage sensor-targeted KCNQ channel activators, which highlight that subtypes of this drug class operate with distinct mechanisms.
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http://dx.doi.org/10.1085/jgp.201812070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279353PMC
December 2018

Slc7a5 regulates Kv1.2 channels and modifies functional outcomes of epilepsy-linked channel mutations.

Nat Commun 2018 10 24;9(1):4417. Epub 2018 Oct 24.

Department of Pharmacology, University of Alberta, Edmonton, T6G 2R3, Canada.

Kv1.2 is a prominent voltage-gated potassium channel that influences action potential generation and propagation in the central nervous system. We explored multi-protein complexes containing Kv1.2 using mass spectrometry followed by screening for effects on Kv1.2. We report that Slc7a5, a neutral amino acid transporter, has a profound impact on Kv1.2. Co-expression with Slc7a5 reduces total Kv1.2 protein, and dramatically hyperpolarizes the voltage-dependence of activation by -47 mV. These effects are attenuated by expression of Slc3a2, a known binding partner of Slc7a5. The profound Slc7a5-mediated current suppression is partly explained by a combination of gating effects including accelerated inactivation and a hyperpolarizing shift of channel activation, causing channels to accumulate in a non-conducting state. Two recently reported Slc7a5 mutations linked to neurodevelopmental delay exhibit a localization defect and have attenuated effects on Kv1.2. In addition, epilepsy-linked gain-of-function Kv1.2 mutants exhibit enhanced sensitivity to Slc7a5.
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http://dx.doi.org/10.1038/s41467-018-06859-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200743PMC
October 2018

One drug-sensitive subunit is sufficient for a near-maximal retigabine effect in KCNQ channels.

J Gen Physiol 2018 10 30;150(10):1421-1431. Epub 2018 Aug 30.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada

Retigabine is an antiepileptic drug and the first voltage-gated potassium (Kv) channel opener to be approved for human therapeutic use. Retigabine is thought to interact with a conserved Trp side chain in the pore of KCNQ2-5 (Kv7.2-7.5) channels, causing a pronounced hyperpolarizing shift in the voltage dependence of activation. In this study, we investigate the functional stoichiometry of retigabine actions by manipulating the number of retigabine-sensitive subunits in concatenated KCNQ3 channel tetramers. We demonstrate that intermediate retigabine concentrations cause channels to exhibit biphasic conductance-voltage relationships rather than progressive concentration-dependent shifts. This suggests that retigabine can exert its effects in a nearly "all-or-none" manner, such that channels exhibit either fully shifted or unshifted behavior. Supporting this notion, concatenated channels containing only a single retigabine-sensitive subunit exhibit a nearly maximal retigabine effect. Also, rapid solution exchange experiments reveal delayed kinetics during channel closure, as retigabine dissociates from channels with multiple drug-sensitive subunits. Collectively, these data suggest that a single retigabine-sensitive subunit can generate a large shift of the KCNQ3 conductance-voltage relationship. In a companion study (Wang et al. 2018. https://doi.org/10.1085/jgp.201812014), we contrast these findings with the stoichiometry of a voltage sensor-targeted KCNQ channel opener (ICA-069673), which requires four drug-sensitive subunits for maximal effect.
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http://dx.doi.org/10.1085/jgp.201812013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168243PMC
October 2018

Four drug-sensitive subunits are required for maximal effect of a voltage sensor-targeted KCNQ opener.

J Gen Physiol 2018 10 30;150(10):1432-1443. Epub 2018 Aug 30.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada

KCNQ2-5 (Kv7.2-Kv7.5) channels are strongly influenced by an emerging class of small-molecule channel activators. Retigabine is the prototypical KCNQ activator that is thought to bind within the pore. It requires the presence of a Trp side chain that is conserved among retigabine-sensitive channels but absent in the retigabine-insensitive KCNQ1 subtype. Recent work has demonstrated that certain KCNQ openers are insensitive to mutations of this conserved Trp, and that their effects are instead abolished or attenuated by mutations in the voltage-sensing domain (VSD). In this study, we investigate the stoichiometry of a VSD-targeted KCNQ2 channel activator, ICA-069673, by forming concatenated channel constructs with varying numbers of drug-insensitive subunits. In homomeric WT KCNQ2 channels, ICA-069673 strongly stabilizes an activated channel conformation, which is reflected in the pronounced deceleration of deactivation and leftward shift of the conductance-voltage relationship. A full complement of four drug-sensitive subunits is required for maximal sensitivity to ICA-069673-even a single drug-insensitive subunit leads to significantly weakened effects. In a companion article (see Yau et al. in this issue), we demonstrate very different stoichiometry for the action of retigabine on KCNQ3, for which a single retigabine-sensitive subunit enables near-maximal effect. Together, these studies highlight fundamental differences in the site and mechanism of activation between retigabine and voltage sensor-targeted KCNQ openers.
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http://dx.doi.org/10.1085/jgp.201812014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168237PMC
October 2018

Probing the molecular basis of hERG drug block with unnatural amino acids.

Sci Rep 2018 01 10;8(1):289. Epub 2018 Jan 10.

Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.

Repolarization of the cardiac action potential is primarily mediated by two voltage-dependent potassium currents: I and I . The voltage-gated potassium channel that gives rise to I , K11.1 (hERG), is uniquely susceptible to high-affinity block by a wide range of drug classes. Pore residues Tyr652 and Phe656 are critical to potent drug interaction with hERG. It is considered that the molecular basis of this broad-spectrum drug block phenomenon occurs through interactions specific to the aromatic nature of the side chains at Tyr652 and Phe656. In this study, we used nonsense suppression to incorporate singly and doubly fluorinated phenylalanine residues at Tyr652 and Phe656 to assess cation-π interactions in hERG terfenadine, quinidine, and dofetilide block. Incorporation of these unnatural amino acids was achieved with minimal alteration to channel activation or inactivation gating. Our assessment of terfenadine, quinidine, and dofetilide block did not reveal evidence of a cation-π interaction at either aromatic residue, but, interestingly, shows that certain fluoro-Phe substitutions at position 652 result in weaker  drug potency.
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http://dx.doi.org/10.1038/s41598-017-18448-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762913PMC
January 2018

PIP2 mediates functional coupling and pharmacology of neuronal KCNQ channels.

Proc Natl Acad Sci U S A 2017 11 23;114(45):E9702-E9711. Epub 2017 Oct 23.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada T6G 2R3;

Retigabine (RTG) is a first-in-class antiepileptic drug that suppresses neuronal excitability through the activation of voltage-gated KCNQ2-5 potassium channels. Retigabine binds to the pore-forming domain, causing a hyperpolarizing shift in the voltage dependence of channel activation. To elucidate how the retigabine binding site is coupled to changes in voltage sensing, we used voltage-clamp fluorometry to track conformational changes of the KCNQ3 voltage-sensing domains (VSDs) in response to voltage, retigabine, and PIP2. Steady-state ionic conductance and voltage sensor fluorescence closely overlap under basal PIP2 conditions. Retigabine stabilizes the conducting conformation of the pore and the activated voltage sensor conformation, leading to dramatic deceleration of current and fluorescence deactivation, but these effects are attenuated upon disruption of channel:PIP2 interactions. These findings reveal an important role for PIP2 in coupling retigabine binding to altered VSD function. We identify a polybasic motif in the proximal C terminus of retigabine-sensitive KCNQ channels that contributes to VSD-pore coupling via PIP2, and thereby influences the unique gating effects of retigabine.
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http://dx.doi.org/10.1073/pnas.1705802114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5692535PMC
November 2017

Site-Directed Unnatural Amino Acid Mutagenesis to Investigate Potassium Channel Pharmacology in Xenopus laevis Oocytes.

Methods Mol Biol 2018 ;1684:253-263

Department of Pharmacology, University of Alberta, 9-70 Medical Sciences Building, Edmonton, AB, Canada, T6G2H7.

Unnatural amino acid mutagenesis is a useful tool enabling detailed investigation of ion channel structure-function relationships and pharmacology. Methods have been developed to apply this technique to different heterologous systems for electrophysiological studies, with each system offering unique advantages and limitations. Synthesis of aminoacylated-tRNA followed by injection into Xenopus laevis oocytes is beneficial because it allows for the incorporation of a wide range of unnatural sidechains, including amino acids with subtle structural differences. Here, we describe a protocol for unnatural amino acid mutagenesis implemented in our lab to study the pharmacology of KCNQ voltage-gated potassium channel opener compounds. This protocol should be applicable to other ion channels and receptor types amenable for functional studies in Xenopus laevis oocytes.
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http://dx.doi.org/10.1007/978-1-4939-7362-0_19DOI Listing
July 2018

Extracellular redox sensitivity of Kv1.2 potassium channels.

Sci Rep 2017 08 22;7(1):9142. Epub 2017 Aug 22.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada.

Kv1.2 is a prominent potassium channel subtype in the nervous system and serves as an important structural template for investigation of ion channel function. However, Kv1.2 voltage-dependence exhibits dramatic cell-to-cell variability due to a gating mode shift that is regulated by an unknown mechanism. We report that this variable behavior is regulated by the extracellular redox environment. Exposure to reducing agents promotes a shift in gating properties towards an 'inhibited' gating mode that resists opening, and causes channels to exhibit pronounced use-dependent activation during trains of repetitive depolarizations. This sensitivity to extracellular redox potential is absent in other Kv1 channels, but is apparent in heteromeric channels containing Kv1.2 subunits, and overlaps with the reported physiological range of extracellular redox couples. Mutagenesis of candidate cysteine residues fails to abolish redox sensitivity. Therefore, we suggest that an extrinsic, redox-sensitive binding partner imparts these properties.
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http://dx.doi.org/10.1038/s41598-017-08718-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567313PMC
August 2017

KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting.

Nat Commun 2016 10 12;7:12795. Epub 2016 Oct 12.

Department of Cardiovascular Medicine, Institute for Genetics of Heart Diseases (IfGH), University Hospital Muenster, Muenster D-48149, Germany.

Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.
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http://dx.doi.org/10.1038/ncomms12795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064022PMC
October 2016

Emerging complexities of lipid regulation of potassium channels.

Authors:
Harley T Kurata

J Gen Physiol 2016 09;148(3):201-5

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

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http://dx.doi.org/10.1085/jgp.201611671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004341PMC
September 2016

Sequence determinants of subtype-specific actions of KCNQ channel openers.

J Physiol 2017 02 23;595(3):663-676. Epub 2016 Sep 23.

Department of Pharmacology, Alberta Diabetes Institute, University of Alberta, Edmonton, AB, Canada.

Key Points: Retigabine is a KCNQ voltage-gated potassium channel opener that was recently approved as an add-on therapeutic for patients with drug-resistant epilepsy. Retigabine exhibits very little specificity between most KCNQ channel subtypes, and there is interest in generating more potent and specific KCNQ channel openers. The present study describes the marked specificity of ICA069673 for KCNQ2 vs. KCNQ3, and exploits this property to investigate determinants of KCNQ subtype specificity. ICA069673 acts on a binding site in the voltage-sensing domain that is distinct from the putative retigabine site in the channel pore. ICA069673 has two separable effects on KCNQ channel activity. We identify two channel residues required for subtype specificity of KCNQ channel openers and show that these are sufficient to generate ICA069673 sensitivity in KCNQ3.

Abstract: Retigabine (RTG) is the first approved anti-epileptic drug that acts via activation of voltage-gated potassium channels, targeting KCNQ channels that underlie the neuronal M-current. RTG exhibits little specificity between KCNQ2-5 as a result of conservation of a Trp residue in the pore domain that binds to the drug. The RTG analogue ICA-069673 ('ICA73') exhibits much stronger effects on KCNQ2 channels, including a large hyperpolarizing shift of the voltage-dependence of activation, an ∼2-fold enhancement of peak current and pronounced subtype specificity for KCNQ2 over KCNQ3. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1-S4) rather than the pore region targeted by RTG. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues F168 and A181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73-mediated gating shift, but preserve RTG sensitivity. Interestingly, A181P mutant channels show little ICA73-mediated gating shift but retain current potentiation by the drug. Mutations (L198F and P211A), which introduce these critical KCNQ2 residues at corresponding positions in KCNQ3, transplant partial ICA73 sensitivity. These findings demonstrate that RTG and ICA73 act via distinct mechanisms, and also reveal specific residues that underlie subtype specificity of KCNQ channel openers.
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http://dx.doi.org/10.1113/JP272762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5285613PMC
February 2017

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels.

Channels (Austin) 2016 8;10(2):158-66. Epub 2015 Dec 8.

b Department of Pharmacology , University of Alberta , Edmonton , AB , Canada.

Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.
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http://dx.doi.org/10.1080/19336950.2015.1120390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4960988PMC
December 2016

Atomic basis for therapeutic activation of neuronal potassium channels.

Nat Commun 2015 Sep 3;6:8116. Epub 2015 Sep 3.

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3.

Retigabine is a recently approved anticonvulsant that acts by potentiating neuronal M-current generated by KCNQ2-5 channels, interacting with a conserved Trp residue in the channel pore domain. Using unnatural amino-acid mutagenesis, we subtly altered the properties of this Trp to reveal specific chemical interactions required for retigabine action. Introduction of a non-natural isosteric H-bond-deficient Trp analogue abolishes channel potentiation, indicating that retigabine effects rely strongly on formation of a H-bond with the conserved pore Trp. Supporting this model, substitution with fluorinated Trp analogues, with increased H-bonding propensity, strengthens retigabine potency. In addition, potency of numerous retigabine analogues correlates with the negative electrostatic surface potential of a carbonyl/carbamate oxygen atom present in most KCNQ activators. These findings functionally pinpoint an atomic-scale interaction essential for effects of retigabine and provide stringent constraints that may guide rational improvement of the emerging drug class of KCNQ channel activators.
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http://dx.doi.org/10.1038/ncomms9116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561856PMC
September 2015

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels.

J Biol Chem 2015 Jun 1;290(25):15450-15461. Epub 2015 May 1.

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Electronic address:

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.
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http://dx.doi.org/10.1074/jbc.M114.631960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505460PMC
June 2015

Use-dependent activation of neuronal Kv1.2 channel complexes.

J Neurosci 2015 Feb;35(8):3515-24

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, Canada, V6T 1Z3

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.
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http://dx.doi.org/10.1523/JNEUROSCI.4518-13.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6605548PMC
February 2015

Atom-by-atom engineering of voltage-gated ion channels: magnified insights into function and pharmacology.

J Physiol 2015 Jun 13;593(12):2627-34. Epub 2015 Mar 13.

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, BC, Canada.

Unnatural amino acid incorporation into ion channels has proven to be a valuable approach to interrogate detailed hypotheses arising from atomic resolution structures. In this short review, we provide a brief overview of some of the basic principles and methods for incorporation of unnatural amino acids into proteins. We also review insights into the function and pharmacology of voltage-gated ion channels that have emerged from unnatural amino acid mutagenesis approaches.
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http://dx.doi.org/10.1113/jphysiol.2014.287714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500348PMC
June 2015

Inward rectifiers and their regulation by endogenous polyamines.

Front Physiol 2014 27;5:325. Epub 2014 Aug 27.

Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia Vancouver, BC, Canada.

Inwardly-rectifying potassium (Kir) channels contribute to maintenance of the resting membrane potential and regulation of electrical excitation in many cell types. Strongly rectifying Kir channels exhibit a very steep voltage dependence resulting in silencing of their activity at depolarized membrane voltages. The mechanism underlying this steep voltage dependence is blockade by endogenous polyamines. These small multifunctional, polyvalent metabolites enter the long Kir channel pore from the intracellular side, displacing multiple occupant ions as they migrate to a stable binding site in the transmembrane region of the channel. Numerous structure-function studies have revealed structural elements of Kir channels that determine their susceptibility to polyamine block, and enable the steep voltage dependence of this process. In addition, various channelopathies have been described that result from alteration of the polyamine sensitivity or activity of strongly rectifying channels. The primary focus of this article is to summarize current knowledge of the molecular mechanisms of polyamine block, and provide some perspective on lingering uncertainties related to this physiologically important mechanism of ion channel blockade. We also briefly review some of the important and well understood physiological roles of polyamine sensitive, strongly rectifying Kir channels, primarily of the Kir2 family.
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http://dx.doi.org/10.3389/fphys.2014.00325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145359PMC
September 2014

Asymmetric functional contributions of acidic and aromatic side chains in sodium channel voltage-sensor domains.

J Gen Physiol 2014 May;143(5):645-56

Department of Anesthesiology, Pharmacology and Therapeutics, and 2 Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

Voltage-gated sodium (NaV) channels mediate electrical excitability in animals. Despite strong sequence conservation among the voltage-sensor domains (VSDs) of closely related voltage-gated potassium (KV) and NaV channels, the functional contributions of individual side chains in Nav VSDs remain largely enigmatic. To this end, natural and unnatural side chain substitutions were made in the S2 hydrophobic core (HC), the extracellular negative charge cluster (ENC), and the intracellular negative charge cluster (INC) of the four VSDs of the skeletal muscle sodium channel isoform (NaV1.4). The results show that the highly conserved aromatic side chain constituting the S2 HC makes distinct functional contributions in each of the four NaV domains. No obvious cation-pi interaction exists with nearby S4 charges in any domain, and natural and unnatural mutations at these aromatic sites produce functional phenotypes that are different from those observed previously in Kv VSDs. In contrast, and similar to results obtained with Kv channels, individually neutralizing acidic side chains with synthetic derivatives and with natural amino acid substitutions in the INC had little or no effect on the voltage dependence of activation in any of the four domains. Interestingly, countercharge was found to play an important functional role in the ENC of DI and DII, but not DIII and DIV. These results suggest that electrostatic interactions with S4 gating charges are unlikely in the INC and only relevant in the ENC of DI and DII. Collectively, our data highlight domain-specific functional contributions of highly conserved side chains in NaV VSDs.
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http://dx.doi.org/10.1085/jgp.201311036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4003186PMC
May 2014

Multiparameter screening reveals a role for Na+ channels in cytokine-induced β-cell death.

Mol Endocrinol 2014 Mar 17;28(3):406-17. Epub 2014 Jan 17.

Department of Cellular and Physiological Sciences (Y.H.C.Y., J.D.J.), Department of Anesthesiology, Pharmacology, and Therapeutics (Y.Y.V., H.T.K.), and Department of Biochemistry and Molecular Biology (M.R.), University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

Pancreatic β-cell death plays a role in both type 1 and type 2 diabetes, but clinical treatments that specifically target β-cell survival have not yet been developed. We have recently developed live-cell imaging-based, high-throughput screening methods capable of identifying factors that modulate pancreatic β-cell death, with the hope of finding drugs that can intervene in this process. In the present study, we used a high-content screen and the Prestwick Chemical Library of small molecules to identify drugs that block cell death resulting from exposure to a cocktail of cytotoxic cytokines (25 ng/mL TNF-α, 10 ng/mL IL-1β, and 10 ng/mL IFN-γ). Data analysis with self-organizing maps revealed that 19 drugs had profiles similar to that of the no cytokine condition, indicating protection. Carbamazepine, an antiepileptic Na(+) channel inhibitor, was particularly interesting because Na(+) channels are not generally considered targets for antiapoptotic therapy in diabetes and because the function of these channels in β-cells has not been well studied. We analyzed the expression and characteristics of Na(+) currents in mature β-cells from MIP-GFP mice. We confirmed the dose-dependent protective effects of carbamazepine and another use-dependent Na(+) channel blocker in cytokine-treated mouse islet cells. Carbamazepine down-regulated the proapoptotic and endoplasmic reticulum stress signaling induced by cytokines. Together, these studies point to Na(+) channels as a novel therapeutic target in diabetes.
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http://dx.doi.org/10.1210/me.2013-1257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5414926PMC
March 2014

Hydrogen bonds as molecular timers for slow inactivation in voltage-gated potassium channels.

Elife 2013 Dec 10;2:e01289. Epub 2013 Dec 10.

Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, Canada.

Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the kinetics of this process remain obscure. Using a combination of synthetic amino acid analogs and concatenated channel subunits we establish two H-bonds near the extracellular surface of the channel that endow Kv channels with a mechanism to time the entry into slow inactivation: an intra-subunit H-bond between Asp447 and Trp434 and an inter-subunit H-bond connecting Tyr445 to Thr439. Breaking of either interaction triggers slow inactivation by means of a local disruption in the selectivity filter, while severing the Tyr445-Thr439 H-bond is likely to communicate this conformational change to the adjacent subunit(s). DOI: http://dx.doi.org/10.7554/eLife.01289.001.
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http://dx.doi.org/10.7554/eLife.01289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3852034PMC
December 2013