Structural Basis for Rapid Voltage Dependent Inactivation of HERG Potassium Channels

Abstract The exquisite fine tuning of biological electrical signalling is mediated by variations in the rates of opening and closing of different ion channels(1). In addition to open and closed conformations, ion channels can exist in an inactivated state, which prevents conduction in the presence of a prolonged activating stimulus(2). Human ether-a-go-go related gene (HERG) K+ channels undergo uniquely rapid and voltage dependent inactivation(3-5), which confers upon them a critical role in protecting against cardiac arrhythmias and sudden death(6). Previous structural studies have captured only the open state of the HERG channel(7,8). Here, we have exploited the K+ sensitivity of HERG inactivation to determine structures of both the conductive state and the elusive inactivated state of HERG. We show that hERG inactivation is facilitated by two competing networks of hydrogen bonds behind the selectivity filter that enable rapid and voltage dependent flipping of the valine carbonyls in the centre of the selectivity filter. Our data also explains how changes in extracellular K+ affects the distribution between conductive and inactivated states(9,10) and thereby explains why hypokalaemia reduces HERG channel activity thereby increasing the risk of cardiac arrhythmias(11).

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Identification of a putative binding site critical for general anesthetic activation of TRPA1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618144114 ◽

2017 ◽

Vol 114 (14) ◽

pp. 3762-3767 ◽

Cited By ~ 25

Author(s):

Hoai T. Ton ◽

Thieu X. Phan ◽

Ara M. Abramyan ◽

Lei Shi ◽

Gerard P. Ahern

Keyword(s):

Ion Channels ◽

Transient Receptor Potential ◽

Transmembrane Domain ◽

Halogen Bond ◽

Critical Role ◽

Sensory Nerves ◽

Structural Basis ◽

General Anesthetics ◽

General Anesthetic ◽

Potential Binding

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

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Amino acid substitutions in the FXYD motif enhance phospholemman-induced modulation of cardiac L-type calcium channels

AJP Cell Physiology ◽

10.1152/ajpcell.00149.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. C1203-C1211 ◽

Cited By ~ 10

Author(s):

Kai Guo ◽

Xianming Wang ◽

Guofeng Gao ◽

Congxin Huang ◽

Keith S. Elmslie ◽

...

Keyword(s):

Amino Acid ◽

Calcium Channels ◽

Molecular Mechanisms ◽

Channel Gating ◽

Fine Tuning ◽

Amino Acid Substitutions ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Kinetics Of

We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149–1159, 2010). The short 17 amino acid extracellular NH2-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate CaV1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with CaV1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on CaV1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca2+ dynamics in the heart.

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Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1515965112 ◽

2015 ◽

Vol 112 (50) ◽

pp. 15366-15371 ◽

Cited By ~ 8

Author(s):

Joshua B. Brettmann ◽

Darya Urusova ◽

Marco Tonelli ◽

Jonathan R. Silva ◽

Katherine A. Henzler-Wildman

Keyword(s):

Ion Selectivity ◽

Selectivity Filter ◽

Structural Basis ◽

Functional Coupling ◽

Dynamic Coupling ◽

Allosteric Coupling ◽

Allosteric Communication ◽

Dependent Inactivation ◽

Ion Binding Sites ◽

Essential Connection

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

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A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin

Science Advances ◽

10.1126/sciadv.abd6922 ◽

2020 ◽

Vol 6 (51) ◽

pp. eabd6922

Author(s):

Maya Lipinsky ◽

William Sam Tobelaim ◽

Asher Peretz ◽

Luba Simhaev ◽

Adva Yeheskel ◽

...

Keyword(s):

Family Member ◽

Selectivity Filter ◽

Wild Type ◽

Voltage Gated ◽

Kv Channel ◽

Kv Channels ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Inactivation Gating ◽

Unique Mechanism

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

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Ion channels and ion selectivity

Essays in Biochemistry ◽

10.1042/ebc20160074 ◽

2017 ◽

Vol 61 (2) ◽

pp. 201-209 ◽

Cited By ~ 31

Author(s):

Benoît Roux

Keyword(s):

Ion Channels ◽

Lipid Membrane ◽

Ion Selectivity ◽

Transport Systems ◽

K Channel ◽

Diffusion Limit ◽

Macromolecular Transport ◽

Loosely Coupled ◽

Voltage Dependent ◽

Opening And Closing

Specific macromolecular transport systems, ion channels and pumps, provide the pathways to facilitate and control the passage of ions across the lipid membrane. Ion channels provide energetically favourable passage for ions to diffuse rapidly and passively according to their electrochemical potential. Selective ion channels are essential for the excitability of biological membranes: the action potential is a transient phenomenon that reflects the rapid opening and closing of voltage-dependent Na+-selective and K+-selective channels. One of the most critical functional aspects of K+ channels is their ability to remain highly selective for K+ over Na+ while allowing high-throughput ion conduction at a rate close to the diffusion limit. Permeation through the K+ channel selectivity filter is believed to proceed as a ‘knockon’ mechanism, in which 2–3 K+ ions interspersed by water molecules move in a single file. Permeation through the comparatively wider and less selective Na+ channels also proceeds via a loosely coupled knockon mechanism, although the ions do not need to be fully dehydrated. While simple structural concepts are often invoked to rationalize the mechanism of ion selectivity, a deeper analysis shows that subtle effects play an important role in these flexible dynamical structures.

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Voltage-Dependent Inactivation Gating at the Selectivity Filter of the MthK K+ channel

Biophysical Journal ◽

10.1016/j.bpj.2010.12.3364 ◽

2011 ◽

Vol 100 (3) ◽

pp. 582a

Author(s):

Andrew S. Thomson ◽

Brad S. Rothberg

Keyword(s):

Selectivity Filter ◽

K Channel ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Inactivation Gating

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Molecular endpoints of Ca2+/calmodulin- and voltage-dependent inactivation of Cav1.3 channels

The Journal of General Physiology ◽

10.1085/jgp.200910308 ◽

2010 ◽

Vol 135 (3) ◽

pp. 197-215 ◽

Cited By ~ 36

Author(s):

Michael R. Tadross ◽

Manu Ben Johny ◽

David T. Yue

Keyword(s):

Selectivity Filter ◽

Mutant Library ◽

Allosteric Inhibition ◽

Channel Activation ◽

Channel Modulation ◽

Dependent Inactivation ◽

Activation Gate ◽

Voltage Dependent ◽

Novel Theory

Ca2+/calmodulin- and voltage-dependent inactivation (CDI and VDI) comprise vital prototypes of Ca2+ channel modulation, rich with biological consequences. Although the events initiating CDI and VDI are known, their downstream mechanisms have eluded consensus. Competing proposals include hinged-lid occlusion of channels, selectivity filter collapse, and allosteric inhibition of the activation gate. Here, novel theory predicts that perturbations of channel activation should alter inactivation in distinctive ways, depending on which hypothesis holds true. Thus, we systematically mutate the activation gate, formed by all S6 segments within CaV1.3. These channels feature robust baseline CDI, and the resulting mutant library exhibits significant diversity of activation, CDI, and VDI. For CDI, a clear and previously unreported pattern emerges: activation-enhancing mutations proportionately weaken inactivation. This outcome substantiates an allosteric CDI mechanism. For VDI, the data implicate a “hinged lid–shield” mechanism, similar to a hinged-lid process, with a previously unrecognized feature. Namely, we detect a “shield” in CaV1.3 channels that is specialized to repel lid closure. These findings reveal long-sought downstream mechanisms of inactivation and may furnish a framework for the understanding of Ca2+ channelopathies involving S6 mutations.

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Analysis of Toxin-Induced Changes in Action Potential Shape for Drug Development

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057109348378 ◽

2009 ◽

Vol 14 (10) ◽

pp. 1228-1235 ◽

Cited By ~ 7

Author(s):

Nesar Akanda ◽

Peter Molnar ◽

Maria Stancescu ◽

James J. Hickman

Keyword(s):

Ion Channels ◽

Action Potential ◽

Shape Analysis ◽

Drug Effects ◽

Model Parameters ◽

Cellular Processes ◽

Potential Shape ◽

Detection And Identification ◽

Voltage Dependent ◽

Opening And Closing

The generation of an action potential (AP) is a complex process in excitable cells that involves the temporal opening and closing of several voltage-dependent ion channels within the cell membrane. The shape of an AP can carry information concerning the state of the involved ion channels as well as their relationship to cellular processes. Alteration of these ion channels by the administration of toxins, drugs, and biochemicals can change the AP’s shape in a specific way, which can be characteristic for a given compound. Thus, AP shape analysis could be a valuable tool for toxin classification and the measurement of drug effects based on their mechanism of action. In an effort to begin classifying the effect of toxins on the shape of intracellularly recorded APs, patch-clamp experiments were performed on NG108-15 hybrid cells in the presence of veratridine, tetraethylammonium, and quinine. To analyze the effect, the authors generated a computer model of the AP mechanism to determine to what extent each ion channel was affected during compound administration based on the changes in the model parameters. This work is a first step toward establishing a new assay system for toxin detection and identification by AP shape analysis.

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Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

The Journal of General Physiology ◽

10.1085/jgp.201010507 ◽

2010 ◽

Vol 136 (5) ◽

pp. 569-579 ◽

Cited By ~ 25

Author(s):

Andrew S. Thomson ◽

Brad S. Rothberg

Keyword(s):

Voltage Sensor ◽

Selectivity Filter ◽

K Channel ◽

K Channels ◽

Dependent Inactivation ◽

Wide Range ◽

Voltage Dependent ◽

Inactivation Gating ◽

External K ◽

Conductive State

Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel’s selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ > Cs+ > Na+ > Li+ ≈ NMG+. Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K+-sensitive inactivation gating, a property that may be common to other K+ channels.

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Fine tuning of neuronal electrical activity: modulation of several ion channels by intracellular messengers in a single identified nerve cell

Journal of Experimental Biology ◽

10.1242/jeb.124.1.307 ◽

1986 ◽

Vol 124 (1) ◽

pp. 307-322

Author(s):

D. P. Lotshaw ◽

E. S. Levitan ◽

I. B. Levitan

Keyword(s):

Ion Channels ◽

Cyclic Amp ◽

Electrical Activity ◽

Nerve Cell ◽

Calcium Current ◽

Fine Tuning ◽

Regulatory Pathways ◽

Intracellular Messengers ◽

Voltage Dependent ◽

Neuronal Electrical Activity

The identified neurone R15 in the abdominal ganglion of the marine mollusc, Aplysia californica, exhibits a rhythmic bursting pattern of electrical activity. This pattern, which is generated endogenously by the interaction of several voltage- and time-dependent ion currents in R15's membrane, is subject to long-term modulation by synaptic stimulation and application of several neurotransmitters. At micromolar concentrations the transmitter serotonin causes neurone R15 to hyperpolarize, as a result of the activation of an anomalously rectifying potassium conductance. Furthermore under some conditions serotonin can excite R15, as a result of the activation of a voltage-dependent calcium current. Both of these effects of serotonin are mediated by the intracellular second messenger cyclic AMP. In addition, serotonin can modulate a chloride current by a cyclic-AMP-dependent mechanism. In contrast to the activation of the voltage-dependent calcium current by serotonin/cyclic AMP, a cyclic GMP analogue alters the bursting pattern by inhibiting this current. The results indicate that a single neurotransmitter, acting via a single intracellular messenger, can modulate several classes of ion channels in a single nerve cell. Furthermore a single class of ion channel, that is responsible for a voltage-dependent calcium current, may be the target for modulation by at least two different intracellular messengers. These findings emphasize the intricacy of the regulatory pathways which contribute to fine tuning of neuronal electrical activity.

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