scholarly journals Integrated Allosteric Model of Voltage Gating of Hcn Channels

2001 ◽  
Vol 117 (6) ◽  
pp. 519-532 ◽  
Author(s):  
Claudia Altomare ◽  
Annalisa Bucchi ◽  
Eva Camatini ◽  
Mirko Baruscotti ◽  
Carlo Viscomi ◽  
...  
Keyword(s):  
Rate Constants ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Hcn Channels ◽  
Open Probability ◽  
Voltage Sensors ◽  
Negative Voltage ◽  
Kinetics Of ◽  
Allosteric Model ◽  
Activation Delay

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation “delay” by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between “reluctant” and “willing” states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

scholarly journals An allosteric model of the molecular interactions of excitation-contraction coupling in skeletal muscle.

1993 ◽  
Vol 102 (3) ◽  
pp. 449-481 ◽  
Author(s):  
E Ríos ◽  
M Karhanek ◽  
J Ma ◽  
A González
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Kinetic Equations ◽  
Voltage Sensor ◽  
Charge Movement ◽  
Calcium Release Channel ◽  
Open Probability ◽  
Release Channel ◽  
Voltage Sensors ◽  
Function Relationship

A contact interaction is proposed to exist between the voltage sensor of the transverse tubular membrane of skeletal muscle and the calcium release channel of the sarcoplasmic reticulum. This interaction is given a quantitative formulation inspired in the Monod, Wyman, and Changeux model of allosteric transitions in hemoglobin (Monod, J., J. Wyman, and J.-P. Changeux. 1965. Journal of Molecular Biology. 12:88-118), and analogous to one proposed by Marks and Jones for voltage-dependent Ca channels (Marks, T. N., and S. W. Jones. 1992. Journal of General Physiology. 99:367-390). The allosteric protein is the calcium release channel, a homotetramer, with two accessible states, closed and open. The kinetics and equilibrium of this transition are modulated by voltage sensors (dihydropyridine receptors) pictured as four units per release channel, each undergoing independent voltage-driven transitions between two states (resting and activating). For each voltage sensor that moves to the activating state, the tendency of the channel to open increases by an equal (large) factor. The equilibrium and kinetic equations of the model are solved and shown to reproduce well a number of experimentally measured relationships including: charge movement (Q) vs. voltage, open probability of the release channel (Po) vs. voltage, the transfer function relationship Po vs. Q, and the kinetics of charge movement, release activation, and deactivation. The main consequence of the assumption of allosteric coupling is that primary effects on the release channel are transmitted backward to the voltage sensor and give secondary effects. Thus, the model reproduces well the effects of perchlorate, described in the two previous articles, under the assumption that the primary effect is to increase the intrinsic tendency of the release channel to open, with no direct effects on the voltage sensor. This modification of the open-closed equilibrium of the release channel causes a shift in the equilibrium dependency of charge movement with voltage. The paradoxical slowing of charge movement by perchlorate also results from reciprocal effects of the channel on the allosterically coupled voltage sensors. The observations of the previous articles plus the simulations in this article constitute functional evidence of allosteric transmission.

scholarly journals The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

2019 ◽  
Author(s):  
Gucan Dai ◽  
Teresa K. Aman ◽  
Frank DiMaio ◽  
William N. Zagotta
Keyword(s):  
Ion Channels ◽  
Metal Ion ◽  
Voltage Sensor ◽  
The Body ◽  
Resting Membrane Potential ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Transmembrane Voltage

Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body1. They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells1. In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration2. VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore3–6. These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)3 and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization7. HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization8–10. The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels11, no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

scholarly journals Mg2+ Enhances Voltage Sensor/Gate Coupling in BK Channels

2007 ◽  
Vol 131 (1) ◽  
pp. 13-32 ◽  
Author(s):  
Frank T. Horrigan ◽  
Zhongming Ma
Keyword(s):  
Resting State ◽  
Binding Sites ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Detectable Effect ◽  
Open Probability ◽  
Independent Manner ◽  
Voltage Sensors ◽  
Channel Opening ◽  
Sensor Activation

BK (Slo1) potassium channels are activated by millimolar intracellular Mg2+ as well as micromolar Ca2+ and membrane depolarization. Mg2+ and Ca2+ act in an approximately additive manner at different binding sites to shift the conductance–voltage (GK-V) relation, suggesting that these ligands might work through functionally similar but independent mechanisms. However, we find that the mechanism of Mg2+ action is highly dependent on voltage sensor activation and therefore differs fundamentally from that of Ca2+. Evidence that Ca2+ acts independently of voltage sensor activation includes an ability to increase open probability (PO) at extreme negative voltages where voltage sensors are in the resting state; 2 μM Ca2+ increases PO more than 15-fold at −120 mV. However 10 mM Mg2+, which has an effect on the GK-V relation similar to 2 μM Ca2+, has no detectable effect on PO when voltage sensors are in the resting state. Gating currents are only slightly altered by Mg2+ when channels are closed, indicating that Mg2+ does not act merely to promote voltage sensor activation. Indeed, channel opening is facilitated in a voltage-independent manner by Mg2+ in a mutant (R210C) whose voltage sensors are constitutively activated. Thus, 10 mM Mg2+ increases PO only when voltage sensors are activated, effectively strengthening the allosteric coupling of voltage sensor activation to channel opening. Increasing Mg2+ from 10 to 100 mM, to occupy very low affinity binding sites, has additional effects on gating that more closely resemble those of Ca2+. The effects of Mg2+ on steady-state activation and IK kinetics are discussed in terms of an allosteric gating scheme and the state-dependent interactions between Mg2+ and voltage sensor that may underlie this mechanism.

scholarly journals IKs ion-channel pore conductance can result from individual voltage sensor movements

2019 ◽  
Vol 116 (16) ◽  
pp. 7879-7888 ◽  
Author(s):  
Maartje Westhoff ◽  
Jodene Eldstrom ◽  
Christopher I. Murray ◽  
Emely Thompson ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Voltage Sensors ◽  
Repolarization Reserve ◽  
Accessory Subunits ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Open States ◽  
Channel Currents

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

scholarly journals Binding of the auxiliary subunit TRIP8b to HCN channels shifts the mode of action of cAMP

2013 ◽  
Vol 142 (6) ◽  
pp. 599-612 ◽  
Author(s):  
Lei Hu ◽  
Bina Santoro ◽  
Andrea Saponaro ◽  
Haiying Liu ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Mechanistic Explanation ◽  
Voltage Gating ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Cation Current ◽  
Auxiliary Subunit ◽  
Channel Opening ◽  
Wide Variability ◽  
The Brain ◽  
Allosteric Model

Hyperpolarization-activated cyclic nucleotide–regulated cation (HCN) channels generate the hyperpolarization-activated cation current Ih present in many neurons. These channels are directly regulated by the binding of cAMP, which both shifts the voltage dependence of HCN channel opening to more positive potentials and increases maximal Ih at extreme negative voltages where voltage gating is complete. Here we report that the HCN channel brain-specific auxiliary subunit TRIP8b produces opposing actions on these two effects of cAMP. In the first action, TRIP8b inhibits the effect of cAMP to shift voltage gating, decreasing both the sensitivity of the channel to cAMP (K1/2) and the efficacy of cAMP (maximal voltage shift); conversely, cAMP binding inhibits these actions of TRIP8b. These mutually antagonistic actions are well described by a cyclic allosteric mechanism in which TRIP8b binding reduces the affinity of the channel for cAMP, with the affinity of the open state for cAMP being reduced to a greater extent than the cAMP affinity of the closed state. In a second apparently independent action, TRIP8b enhances the action of cAMP to increase maximal Ih. This latter effect cannot be explained by the cyclic allosteric model but results from a previously uncharacterized action of TRIP8b to reduce maximal current through the channel in the absence of cAMP. Because the binding of cAMP also antagonizes this second effect of TRIP8b, application of cAMP produces a larger increase in maximal Ih in the presence of TRIP8b than in its absence. These findings may provide a mechanistic explanation for the wide variability in the effects of modulatory transmitters on the voltage gating and maximal amplitude of Ih reported for different neurons in the brain.

scholarly journals Voltage Sensor Movement and cAMP Binding Allosterically Regulate an Inherently Voltage-independent Closed−Open Transition in HCN Channels

2007 ◽  
Vol 129 (2) ◽  
pp. 175-188 ◽  
Author(s):  
Shan Chen ◽  
Jing Wang ◽  
Lei Zhou ◽  
Meena S. George ◽  
Steven A. Siegelbaum
Keyword(s):  
Cyclic Nucleotide ◽  
Voltage Sensor ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Voltage Range ◽  
Slow Kinetics ◽  
Rapid Kinetics ◽  
Hcn1 Channels ◽  
Rate Limiting ◽  
Allosteric Model

The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4–S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed–open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.

scholarly journals Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate

2012 ◽  
Vol 140 (5) ◽  
pp. 469-479 ◽  
Author(s):  
Sujung Ryu ◽  
Gary Yellen
Keyword(s):  
Voltage Dependence ◽  
Voltage Sensor ◽  
Hcn Channels ◽  
Charge Movement ◽  
Coupling Energy ◽  
K Channels ◽  
Voltage Sensors ◽  
Gating Charge ◽  
Pacemaker Channels ◽  
Open States

HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K+ channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K+ channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd2+ ions freeze the open–closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd2+ and measure the effect on voltage sensor movement. Cd2+ did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd2+-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open–closed equilibrium, corresponding to a coupling energy of ∼1.3–2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K+ channels, but also much weaker.

scholarly journals Mg2+ binding to open and closed states can activate BK channels provided that the voltage sensors are elevated

2011 ◽  
Vol 138 (6) ◽  
pp. 593-607 ◽  
Author(s):  
Ren-Shiang Chen ◽  
Yanyan Geng ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Closed Interval ◽  
Open Interval ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Interval Duration ◽  
Open Probability ◽  
Voltage Sensors ◽  
High Affinity ◽  
The Mean

BK channels are activated by intracellular Ca2+ and Mg2+ as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca2+ sites, one low-affinity Mg2+ site, and a voltage sensor. This study further investigates the mechanism of Mg2+ activation by using single-channel recording to determine separately the action of Mg2+ on the open and closed states of the channel. To limit Mg2+ action to the Mg2+ sites, the two high-affinity Ca2+ sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg2+ decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg2+ can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg2+ action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (Po) is dramatically increased by 10 mM Mg2+ when voltage sensors are activated with either depolarization or the mutation R210C. The increased Po arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg2+ has no detectable effects on Po or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg2+ can bind to and alter the gating of both closed and open states to increase Po, provided that one or more voltage sensors are activated.

scholarly journals Coupling between Voltage Sensor Activation, Ca2+ Binding and Channel Opening in Large Conductance (BK) Potassium Channels

2002 ◽  
Vol 120 (3) ◽  
pp. 267-305 ◽  
Author(s):  
Frank T. Horrigan ◽  
Richard W. Aldrich
Keyword(s):  
Steady State ◽  
Conformational Change ◽  
Bk Channels ◽  
Voltage Sensor ◽  
Kinetic Properties ◽  
Open Probability ◽  
Voltage Sensors ◽  
Channel Opening ◽  
Wide Range ◽  
Sensor Activation

To determine how intracellular Ca2+ and membrane voltage regulate the gating of large conductance Ca2+-activated K+ (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca2+ over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305–336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). In 0 Ca2+, the steady-state gating charge-voltage (QSS-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (GK-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 μM Ca2+. This change reflects a differential effect of Ca2+ on voltage sensor activation and channel opening. Ca2+ has only a small effect on the fast component of ON gating current, indicating that Ca2+ binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than −80 mV) increases more than 1,000-fold in 70 μM Ca2+, demonstrating that Ca2+ increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca2+ binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca2+ sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca2+ sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic IK kinetics indicate that Ca2+ and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.

scholarly journals Allosteric Voltage Gating of Potassium Channels II

1999 ◽  
Vol 114 (2) ◽  
pp. 305-336 ◽  
Author(s):  
Frank T. Horrigan ◽  
Richard W. Aldrich
Keyword(s):  
Preceding Paper ◽  
Voltage Sensor ◽  
Voltage Gating ◽  
Total Charge ◽  
Charge Movement ◽  
Gating Currents ◽  
Open Probability ◽  
K Channels ◽  
Virtual Absence ◽  
Gating Charge

Large-conductance Ca2+-activated K+ channels can be activated by membrane voltage in the absence of Ca2+ binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca2+-activated K+ channels in the virtual absence of Ca2+ (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge–voltage relationship (Q–V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G–V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (τON = 60 μs at +200 mV, τOFF = 16 μs at −80 mV). However, QOFF increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of IK activation. The slow onset of this gating charge prevents its detection as a component of IgON, although it represents ∼40% of the total charge moved at +140 mV. The decay of IgOFF is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C–C, O–O, and C–O transitions.

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