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

2010 ◽  
Vol 135 (3) ◽  
pp. 197-215 ◽  
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.

scholarly journals Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel

2018 ◽  
Vol 150 (9) ◽  
pp. 1273-1286 ◽  
Author(s):  
Vinay Idikuda ◽  
Weihua Gao ◽  
Khade Grant ◽  
Zhuocheng Su ◽  
Qinglian Liu ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Molecular Level ◽  
Basic Research ◽  
Laser Pulses ◽  
Oxygen Species ◽  
Dependent Inactivation ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Short Laser Pulses ◽  
Working Distance

Photochemically or metabolically generated singlet oxygen (1O2) reacts broadly with macromolecules in the cell. Because of its short lifetime and working distance, 1O2 holds potential as an effective and precise nanoscale tool for basic research and clinical practice. Here we investigate the modification of the spHCN channel that results from photochemically and chemically generated 1O2. The spHCN channel shows strong voltage-dependent inactivation in the absence of cAMP. In the presence of photosensitizers, short laser pulses transform the gating properties of spHCN by abolishing inactivation and increasing the macroscopic current amplitude. Alanine replacement of a histidine residue near the activation gate within the channel’s pore abolishes key modification effects. Application of a variety of chemicals including 1O2 scavengers and 1O2 generators supports the involvement of 1O2 and excludes other reactive oxygen species. This study provides new understanding about the photodynamic modification of ion channels by 1O2 at the molecular level.

scholarly journals Structural basis of ion permeation gating in Slo2.1 K+ channels

2013 ◽  
Vol 142 (5) ◽  
pp. 523-542 ◽  
Author(s):  
Priyanka Garg ◽  
Alison Gardner ◽  
Vivek Garg ◽  
Michael C. Sanguinetti
Keyword(s):  
Selectivity Filter ◽  
Structural Basis ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Ion Permeation ◽  
Channel Activation ◽  
K Channels ◽  
Voltage Gated ◽  
Channel Function ◽  
Activation Gate

The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K+ channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K+ channel that is activated by intracellular Na+ and Cl−. Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation.

scholarly journals Retigabine: Bending Potassium Channels to Our Will

2005 ◽  
Vol 5 (5) ◽  
pp. 166-168 ◽  
Author(s):  
Andre Lagrange
Keyword(s):  
Anticonvulsant Drug ◽  
Structural Determinants ◽  
Channel Activation ◽  
Knock Out ◽  
Strong Shift ◽  
Type K ◽  
Channel Opening ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Hyperpolarizing Shift

The New Anticonvulsant Retigabine Favors Voltage-dependent Opening of the Kv7.2 (KCNQ2) Channel by Binding to Its Activation Gate Wuttke TV, Seebohm G, Bail S, Maljevic S, Lerche H Mol Pharmacol 2005;67:1009–1017 Retigabine (RTG) is an anticonvulsant drug with a novel mechanism of action. It activates neuronal KCNQ-type K+ channels by inducing a large hyperpolarizing shift of steady-state activation. To identify the structural determinants of KCNQ channel activation by RTG, we constructed a set of chimeras by using the neuronal KV7.2 ( KCNQ2) channel, which is activated by RTG, and the cardiac KV7.1 ( KCNQ1) channel, which is not affected by this drug. Substitution of either the S5 or the S6 segment in KV7.2 by the respective parts of KV7.1 led to a complete loss of activation by RTG. Trp236 in the cytoplasmic part of S5 and the conserved Gly301 in S6 (KV7.2), considered as the gating hinge (Ala336 in KV7.1), were found to be crucial for the RTG effect: mutation of these residues could either knock out the effect in KV7.2 or restore it partially in KV7.1/KV7.2 chimeras. We propose that RTG binds to a hydrophobic pocket formed upon channel opening between the cytoplasmic parts of S5 and S6 involving Trp236 and the channel's gate, which could well explain the strong shift in voltage-dependent activation.

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

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.

scholarly journals Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Jing Li ◽  
Rong Shen ◽  
Ahmed Rohaim ◽  
Ramon Mendoza Uriarte ◽  
Mikolai Fajer ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Computational Study ◽  
Homology Model ◽  
Selectivity Filter ◽  
K Channels ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Signature Sequence ◽  
Dynamics Simulations ◽  
Pore Domain

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

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

2010 ◽  
Vol 136 (5) ◽  
pp. 569-579 ◽  
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.

scholarly journals Structural Basis for Rapid Voltage Dependent Inactivation of HERG Potassium Channels

2021 ◽  
Author(s):  
Jamie Vandenberg ◽  
Carus Lau ◽  
Emelie Flood ◽  
Mark Hunter ◽  
Chai-Ann Ng ◽  
...  
Keyword(s):  
Ion Channels ◽  
Cardiac Arrhythmias ◽  
Critical Role ◽  
Selectivity Filter ◽  
Fine Tuning ◽  
Herg Channel ◽  
Structural Basis ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Opening And Closing

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).

scholarly journals Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate.

2021 ◽  
Author(s):  
Bethan A Cole ◽  
Nadia Pilati ◽  
Jonathan D Lippiat
Keyword(s):  
Intracellular Sodium ◽  
Selectivity Filter ◽  
Channel Structure ◽  
Channel Activity ◽  
Gain Of Function ◽  
Activation Kinetics ◽  
Pathogenic Variants ◽  
Activation Gate ◽  
Sodium Dependent ◽  
Voltage Dependent

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

scholarly journals Activation-inactivation of potassium channels and development of the potassium-channel spike in internally perfused squid giant axons.

1981 ◽  
Vol 78 (1) ◽  
pp. 43-61 ◽  
Author(s):  
I Inoue
Keyword(s):  
Potassium Channels ◽  
Potassium Channel ◽  
Voltage Clamp ◽  
Time Constant ◽  
Equilibrium Potential ◽  
Giant Axons ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Time Courses ◽  
Calcium Permeability

A spike that is the result of calcium permeability through potassium channels was separated from the action potential is squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution by blocking sodium channels with tetrodotoxin. Currents through potassium channels were studied under voltage clamp. The records showed a clear voltage-dependent inactivation of the currents. The inactivation was composed of at least two components; one relatively fast, having a time constant of 20--30 ms, and the other very slow, having a time constant of 5--10 s. Voltage clamp was carried out with a variety of salt compositions in both the internal and external solutions. A similar voltage-dependent inactivation, also composed of the two components, was recognized in all the current through potassium channels. Although the direction and intensity of current strongly depended on the salt composition of the solutions, the time-courses of these currents at corresponding voltages were very similar. These results strongly suggest that the inactivation of the currents in attributable to an essential, dynamic property of potassium channels themselves. Thus, the generation of a potassium-channel spike can be understood as an event that occurs when the equilibrium potential across the potassium channel becomes positive.

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