scholarly journals Status of the Intracellular Gate in the Activated-not-open State of Shaker K+ Channels

2005 ◽  
Vol 126 (5) ◽  
pp. 419-428 ◽  
Author(s):  
Donato del Camino ◽  
Max Kanevsky ◽  
Gary Yellen
Keyword(s):  
Conformational Changes ◽  
Charge Movement ◽  
Ion Flow ◽  
K Channels ◽  
Closed State ◽  
Channel Opening ◽  
Activated State ◽  
Form Part ◽  
Voltage Dependent ◽  
Flow Through

Voltage-dependent K+ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd2+ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.

scholarly journals Calmodulin acts as a state-dependent switch to control a cardiac potassium channel opening

2020 ◽  
Vol 6 (50) ◽  
pp. eabd6798
Author(s):  
Po Wei Kang ◽  
Annie M. Westerlund ◽  
Jingyi Shi ◽  
Kelli McFarland White ◽  
Alex K. Dou ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Conformational Changes ◽  
State Dependent ◽  
Gating Mechanism ◽  
Channel Opening ◽  
Activated State ◽  
Voltage Dependent ◽  
Functional Consequences ◽  
Open States ◽  
Voltage Sensing Domain

Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP2) are potent regulators of the voltage-gated potassium channel KCNQ1 (KV7.1), which conducts the cardiac IKs current. Although cryo–electron microscopy structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP2, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully activated state allows PIP2 to compete with CaM for binding to VSD. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. We identify a motif in the KCNQ1 cytosolic domain, which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP2 and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its physiological function.

scholarly journals Hydrophobic gasket mutation produces gating pore currents in closed human voltage-gated proton channels

2019 ◽  
Vol 116 (38) ◽  
pp. 18951-18961 ◽  
Author(s):  
Richard Banh ◽  
Vladimir V. Cherny ◽  
Deri Morgan ◽  
Boris Musset ◽  
Sarah Thomas ◽  
...  
Keyword(s):  
Proton Channel ◽  
Voltage Gated ◽  
Closed State ◽  
Channel Opening ◽  
Proton Current ◽  
Voltage Dependent ◽  
Current Flows ◽  
Voltage Gated Ion Channels ◽  
Dynamics Simulations ◽  
Voltage Sensing Domain

The hydrophobic gasket (HG), a ring of hydrophobic amino acids in the voltage-sensing domain of most voltage-gated ion channels, forms a constriction between internal and external aqueous vestibules. Cationic Arg or Lys side chains lining the S4 helix move through this “gating pore” when the channel opens. S4 movement may occur during gating of the human voltage-gated proton channel, hHV1, but proton current flows through the same pore in open channels. Here, we replaced putative HG residues with less hydrophobic residues or acidic Asp. Substitution of individuals, pairs, or all 3 HG positions did not impair proton selectivity. Evidently, the HG does not act as a secondary selectivity filter. However, 2 unexpected functions of the HG in HV1 were discovered. Mutating HG residues independently accelerated channel opening and compromised the closed state. Mutants exhibited open–closed gating, but strikingly, at negative voltages where “normal” gating produces a nonconducting closed state, the channel leaked protons. Closed-channel proton current was smaller than open-channel current and was inhibited by 10 μM Zn2+. Extreme hyperpolarization produced a deeper closed state through a weakly voltage-dependent transition. We functionally identify the HG as Val109, Phe150, Val177, and Val178, which play a critical and exclusive role in preventing H+ influx through closed channels. Molecular dynamics simulations revealed enhanced mobility of Arg208 in mutants exhibiting H+ leak. Mutation of HG residues produces gating pore currents reminiscent of several channelopathies.

scholarly journals Conformational Changes in the Pore of CLC-0

2003 ◽  
Vol 122 (3) ◽  
pp. 277-294 ◽  
Author(s):  
Alessio Accardi ◽  
Michael Pusch
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
General Structure ◽  
Point Mutations ◽  
Clofibric Acid ◽  
Selectivity Filter ◽  
Side Chain ◽  
Closed State ◽  
State Dependent ◽  
Channel Opening

The Torpedo Cl− channel, CLC-0, is inhibited by clofibric acid derivatives from the intracellular side. We used the slow gate-deficient mutant CLC-0C212S to investigate the mechanism of block by the clofibric acid–derivative p-chlorophenoxy-acetic acid (CPA). CPA blocks open channels with low affinity (KDO= 45 mM at 0 mV) and shows fast dissociation (koff = 490 s−1 at −140 mV). In contrast, the blocker binds to closed channels with higher affinity and with much slower kinetics. This state-dependent block coupled with the voltage dependence of the gating transitions results in a highly voltage-dependent inhibition of macroscopic currents (KD ∼1 mM at −140 mV; KD ∼65 mM at 60 mV). The large difference in CPA affinity of the open and closed state suggests that channel opening involves more than just a local conformational rearrangement. On the other hand, in a recent work (Dutzler, R., E.B. Campbell, and R. MacKinnon. 2003. Science. 300:108–112) it was proposed that the conformational change underlying channel opening is limited to a movement of a single side chain. A prediction of this latter model is that mutations that influence CPA binding to the channel should affect the affinities for an open and closed channel in a similar manner since the general structure of the pore remains largely unchanged. To test this hypothesis we introduced point mutations in four residues (S123, T471, Y512, and K519) that lie close to the intracellular pore mouth or to the putative selectivity filter. Mutation T471S alters CPA binding exclusively to closed channels. Pronounced effects on the open channel block are observed in three other mutants, S123T, Y512A, and K519Q. Together, these results collectively suggest that the structure of the CPA binding site is different in the open and closed state. Finally, replacement of Tyr 512, a residue directly coordinating the central Cl− ion in the crystal structure, with Phe or Ala has very little effect on single channel conductance and selectivity. These observations suggest that channel opening in CLC-0 consists in more than a movement of a side chain and that other parts of the channel and of the selectivity filter are probably involved.

scholarly journals Generalized Anxiety Disorder in Adults: Focus on Pregabalin

2011 ◽  
Vol 3 ◽  
pp. CMPsy.S5069 ◽  
Author(s):  
Mark J. Boschen
Keyword(s):  
Anxiety Disorder ◽  
Generalized Anxiety Disorder ◽  
Preliminary Evidence ◽  
Future Research ◽  
Generalized Anxiety ◽  
Inhibitory Neurotransmitter ◽  
Ion Flow ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels ◽  
Flow Through

Generalized anxiety disorder (GAD) is a chronic illness which impacts significantly on an individual's functioning and quality of life. Pregabalin is a novel structural analogue of the inhibitory neurotransmitter GABA, acting to reduce calcium ion flow through the α2δ subunit of pre-synaptic voltage-dependent calcium channels. Pregabalin has been used in treatment of GAD in a total of eight published controlled trials. In each trial, pregabalin has demonstrated a superiority over placebo, with response rates of over 40% in all studies, including patients on lower doses. One study has provided preliminary evidence for the efficacy of pregabalin in treatment of GAD in older adults. Pregabalin is generally well tolerated, with the most common adverse events being dizziness and somnolence. Adverse effects are generally mild-to-moderate, and transient. Pregabalin has low abuse potential. Limitations of the current literature are discussed, and directions for future research are proposed.

scholarly journals Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels.

1996 ◽  
Vol 108 (3) ◽  
pp. 143-155 ◽  
Author(s):  
F Noceti ◽  
P Baldelli ◽  
X Wei ◽  
N Qin ◽  
L Toro ◽  
...  
Keyword(s):  
Ionic Current ◽  
Variance Analysis ◽  
Beta Subunit ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
K Channels ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Ca2 Channels

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.

scholarly journals Convergence of biological and psychological perspectives on cognitive coordination in schizophrenia

2003 ◽  
Vol 26 (1) ◽  
pp. 65-82 ◽  
Author(s):  
William A. Phillips ◽  
Steven M. Silverstein
Keyword(s):  
Brain Function ◽  
Important Class ◽  
Neural Basis ◽  
Ion Flow ◽  
Control Gain ◽  
Mode Of Operation ◽  
Voltage Dependent ◽  
Nmda Channels ◽  
Flow Through ◽  
Cortical Regions

AbstractThe concept of locally specialized functions dominates research on higher brain function and its disorders. Locally specialized functions must be complemented by processes that coordinate those functions, however, and impairment of coordinating processes may be central to some psychotic conditions. Evidence for processes that coordinate activity is provided by neurobiological and psychological studies of contextual disambiguation and dynamic grouping. Mechanisms by which this important class of cognitive functions could be achieved include those long-range connections within and between cortical regions that activate synaptic channels via NMDA-receptors, and which control gain through their voltage-dependent mode of operation. An impairment of these mechanisms is central to PCP-psychosis, and the cognitive capabilities that they could provide are impaired in some forms of schizophrenia. We conclude that impaired cognitive coordination due to reduced ion flow through NMDA-channels is involved in schizophrenia, and we suggest that it may also be involved in other disorders. This perspective suggests several ways in which further research could enhance our understanding of cognitive coordination, its neural basis, and its relevance to psychopathology.

scholarly journals A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

2017 ◽  
Vol 149 (5) ◽  
pp. 577-593 ◽  
Author(s):  
Adam P. Tomczak ◽  
Jorge Fernández-Trillo ◽  
Shashank Bharill ◽  
Ferenc Papp ◽  
Gyorgy Panyi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Voltage Sensor ◽  
Ion Flow ◽  
Gating Model ◽  
Cryo Electron Microscopy ◽  
Channel Gate ◽  
Voltage Dependent ◽  
Voltage Gated Ion Channels ◽  
Voltage Sensing Domain ◽  
Pore Domain

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4–S5 linker). However, our recent work on channels disrupted in the S4–S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4–S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use “split” channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4–S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4–S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism.

scholarly journals Structural basis for activation of voltage sensor domains in an ion channel TPC1

2018 ◽  
Vol 115 (39) ◽  
pp. E9095-E9104 ◽  
Author(s):  
Alexander F. Kintzer ◽  
Evan M. Green ◽  
Pawel K. Dominik ◽  
Michael Bridges ◽  
Jean-Paul Armache ◽  
...  
Keyword(s):  
Ion Channel ◽  
Conformational Changes ◽  
Electrical Potential ◽  
Structural Basis ◽  
Ion Conductance ◽  
Activated State ◽  
Voltage Dependent ◽  
Internal Side ◽  
Transmembrane Electrical Potential ◽  
Gating Charges

Voltage-sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured an example of a resting-state VSD in an intact ion channel. To generate an activated-state VSD in the same channel we removed the luminal inhibitory Ca2+-binding site (Cai2+), which shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca2+ to a cytoplasmic site (Caa2+). An X-ray structure with Caa2+ removed and a near-atomic resolution cryo-EM structure with Cai2+ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage-dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel’s requirement of cytoplasmic Ca2+ ions for activation.

scholarly journals Closed state-coupled C-type inactivation in BK channels

2016 ◽  
Vol 113 (25) ◽  
pp. 6991-6996 ◽  
Author(s):  
Jiusheng Yan ◽  
Qin Li ◽  
Richard W. Aldrich
Keyword(s):  
Conformational Changes ◽  
Bk Channels ◽  
Bk Channel ◽  
Membrane Potentials ◽  
Selectivity Filter ◽  
Open Probability ◽  
Closed State ◽  
State Dependent ◽  
Channel Opening ◽  
Activation Gate

Ion channels regulate ion flow by opening and closing their pore gates. K+ channels commonly possess two pore gates, one at the intracellular end for fast channel activation/deactivation and the other at the selectivity filter for slow C-type inactivation/recovery. The large-conductance calcium-activated potassium (BK) channel lacks a classic intracellular bundle-crossing activation gate and normally show no C-type inactivation. We hypothesized that the BK channel’s activation gate may spatially overlap or coexist with the C-type inactivation gate at or near the selectivity filter. We induced C-type inactivation in BK channels and studied the relationship between activation/deactivation and C-type inactivation/recovery. We observed prominent slow C-type inactivation/recovery in BK channels by an extreme low concentration of extracellular K+ together with a Y294E/K/Q/S or Y279F mutation whose equivalent in Shaker channels (T449E/K/D/Q/S or W434F) caused a greatly accelerated rate of C-type inactivation or constitutive C-inactivation. C-type inactivation in most K+ channels occurs upon sustained membrane depolarization or channel opening and then recovers during hyperpolarized membrane potentials or channel closure. However, we found that the BK channel C-type inactivation occurred during hyperpolarized membrane potentials or with decreased intracellular calcium ([Ca2+]i) and recovered with depolarized membrane potentials or elevated [Ca2+]i. Constitutively open mutation prevented BK channels from C-type inactivation. We concluded that BK channel C-type inactivation is closed state-dependent and that its extents and rates inversely correlate with channel-open probability. Because C-type inactivation can involve multiple conformational changes at the selectivity filter, we propose that the BK channel’s normal closing may represent an early conformational stage of C-type inactivation.

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