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

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.

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Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating

eLife ◽

10.7554/elife.53400 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 13

Author(s):

Marina A Kasimova ◽

Debanjan Tewari ◽

John B Cowgill ◽

Willy Carrasquel Ursuleaz ◽

Jenna L Lin ◽

...

Keyword(s):

Ion Channels ◽

Voltage Sensor ◽

Inward Rectification ◽

Hcn Channels ◽

Hcn Channel ◽

Functional Studies ◽

Gating Charge ◽

Helical Turn ◽

Voltage Gated Ion Channels ◽

Dynamics Simulations

In contrast to most voltage-gated ion channels, hyperpolarization- and cAMP gated (HCN) ion channels open on hyperpolarization. Structure-function studies show that the voltage-sensor of HCN channels are unique but the mechanisms that determine gating polarity remain poorly understood. All-atom molecular dynamics simulations (~20 μs) of HCN1 channel under hyperpolarization reveals an initial downward movement of the S4 voltage-sensor but following the transfer of last gating charge, the S4 breaks into two sub-helices with the lower sub-helix becoming parallel to the membrane. Functional studies on bipolar channels show that the gating polarity strongly correlates with helical turn propensity of the substituents at the breakpoint. Remarkably, in a proto-HCN background, the replacement of breakpoint serine with a bulky hydrophobic amino acid is sufficient to completely flip the gating polarity from inward to outward-rectifying. Our studies reveal an unexpected mechanism of inward rectification involving a linker sub-helix emerging from HCN S4 during hyperpolarization.

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Structural changes during HCN channel gating defined by high affinity metal bridges

The Journal of General Physiology ◽

10.1085/jgp.201210838 ◽

2012 ◽

Vol 140 (3) ◽

pp. 279-291 ◽

Cited By ~ 26

Author(s):

Daniel C.H. Kwan ◽

David L. Prole ◽

Gary Yellen

Keyword(s):

Structural Changes ◽

Structural Model ◽

Channel Gating ◽

Structural Basis ◽

Hcn Channels ◽

Hcn Channel ◽

Membrane Hyperpolarization ◽

Linker Region ◽

High Affinity ◽

Gating Mechanism

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

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HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

10.1101/2020.03.02.973560 ◽

2020 ◽

Author(s):

Ze-Jun Wang ◽

Ismary Blanco ◽

Sebastien Hayoz ◽

Tinatin I. Brelidze

Keyword(s):

Cyclic Nucleotide ◽

Transmembrane Domain ◽

Rhythmic Activity ◽

Surface Expression ◽

Hcn Channels ◽

Hcn Channel ◽

Structural Element ◽

Functional Link ◽

Membrane Hyperpolarization ◽

Membrane Spanning

ABSTRACTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane domain via the C-linker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage- and cAMP-dependent mechanisms of HCN channel gating.

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HCN channel-mediated neuromodulation can control action potential velocity and fidelity in central axons

eLife ◽

10.7554/elife.42766 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 5

Author(s):

Niklas Byczkowicz ◽

Abdelmoneim Eshra ◽

Jacqueline Montanaro ◽

Andrea Trevisiol ◽

Johannes Hirrlinger ◽

...

Keyword(s):

Action Potential ◽

Conduction Velocity ◽

Mossy Fiber ◽

Mossy Fibers ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Resting Membrane Potential ◽

Immunogold Electron Microscopy ◽

Hcn Channels ◽

Hcn Channel

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical rhythmicity and excitability in the heart and brain, but the function of HCN channels at the subcellular level in axons remains poorly understood. Here, we show that the action potential conduction velocity in both myelinated and unmyelinated central axons can be bidirectionally modulated by a HCN channel blocker, cyclic adenosine monophosphate (cAMP), and neuromodulators. Recordings from mouse cerebellar mossy fiber boutons show that HCN channels ensure reliable high-frequency firing and are strongly modulated by cAMP (EC50 40 µM; estimated endogenous cAMP concentration 13 µM). In addition, immunogold-electron microscopy revealed HCN2 as the dominating subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 channels control conduction velocity primarily by altering the resting membrane potential and are associated with significant metabolic costs. These results suggest that the cAMP-HCN pathway provides neuromodulators with an opportunity to finely tune energy consumption and temporal delays across axons in the brain.

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S4 Movement in a Mammalian HCN Channel

The Journal of General Physiology ◽

10.1085/jgp.200308916 ◽

2003 ◽

Vol 123 (1) ◽

pp. 21-32 ◽

Cited By ~ 61

Author(s):

Sriharsha Vemana ◽

Shilpi Pandey ◽

H. Peter Larsson

Keyword(s):

Voltage Sensor ◽

K Channel ◽

Hcn Channels ◽

Dependent Manner ◽

Hcn Channel ◽

Voltage Range ◽

Kv Channels ◽

State Dependent ◽

Voltage Dependent ◽

Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

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Integrated Allosteric Model of Voltage Gating of Hcn Channels

The Journal of General Physiology ◽

10.1085/jgp.117.6.519 ◽

2001 ◽

Vol 117 (6) ◽

pp. 519-532 ◽

Cited By ~ 105

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.

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Carbon monoxide stimulates a potassium-selective current in rabbit corneal epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1994.267.2.c435 ◽

1994 ◽

Vol 267 (2) ◽

pp. C435-C442 ◽

Cited By ~ 29

Author(s):

A. Rich ◽

G. Farrugia ◽

J. L. Rae

Keyword(s):

Epithelial Cells ◽

Single Channel ◽

Cell Voltage ◽

Resting Membrane Potential ◽

Open Probability ◽

Corneal Epithelial Cells ◽

Membrane Hyperpolarization ◽

Corneal Epithelial ◽

Intracellular Cgmp ◽

Excised Patches

The effects of CO on ion currents in freshly dispersed rabbit corneal epithelial cells were assessed using the perforated patch whole cell voltage-clamp technique. Bath perfusion with 1% CO resulted in a 84 +/- 18% (mean +/- SE, n = 14) increase in potassium current (IK) and a membrane hyperpolarization from -42 +/- 4 to -51 +/- 4 mV. The CO-stimulated current reversed at -64 +/- 7 mV [reverse potential (EK) = -87 mV]. The stimulated current was blocked by 1 mM quinidine or 1 mM diltiazem, agents that inhibit IK in rabbit corneal epithelial cells. Single potassium-channel currents measured in the cell-attached configuration showed that exogenous CO increased the steady-state open probability from 0.003 to 0.156 at a holding potential of -40 mV. CO did not affect open probability in excised patches. The single-channel conductance measured from -40 to +40 mV was unaffected. Intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration measured with radioimmunoassay techniques was found to increase from 0.41 +/- 0.24 to 0.55 +/- 0.27 pmol/10(6) cells after the addition of 1% CO (P < 0.05). The data show that bath perfusion with exogenous CO activates IK and hyperpolarizes the resting membrane potential; the data also suggest that CO modulates intracellular cGMP concentration.

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Distinct Populations of HCN Pacemaker Channels Produce Voltage-dependent and Voltage-independent Currents

The Journal of General Physiology ◽

10.1085/jgp.200509389 ◽

2006 ◽

Vol 127 (2) ◽

pp. 183-190 ◽

Cited By ~ 36

Author(s):

Catherine Proenza ◽

Gary Yellen

Keyword(s):

Channel Blocker ◽

Hcn Channels ◽

Hcn Channel ◽

Single Application ◽

Distinct Population ◽

Open Probability ◽

Pacemaker Channels ◽

Conduction Pathway ◽

Voltage Dependent

Hyperpolarization-activated HCN pacemaker channels are critical for the generation of spontaneous activity and the regulation of excitability in the heart and in many types of neurons. These channels produce both a voltage-dependent current (Ih) and a voltage-independent current (Iinst or VIC). In this study, we explored the molecular basis of the voltage-independent current. We found that for the spHCN isoform, VIC averaged ∼4% of the maximum HCN conductance that could be activated by hyperpolarization. Cyclic AMP increased the voltage-independent current in spHCN to ∼8% of maximum. In HCN2, VIC was ∼2% of the maximal current, and was little affected by cAMP. VIC in both spHCN and HCN2 was blocked rapidly both by ZD7288 (an HCN channel blocker that is thought to bind in the conduction pore) and by application of Cd2+ to channels containing an introduced cysteine in the pore (spHCN-464C or HCN2-436C). These results suggest that VIC flows through the main conduction pathway, down the central axis of the protein. We suspected that VIC simply represented a nonzero limiting open probability for HCN channels at positive voltages. Surprisingly, we found instead that the spHCN channels carrying VIC were not in rapid equilibrium with the channels carrying the voltage-dependent current, because they could be blocked independently; a single application of blocker at a depolarized potential essentially eliminated VIC with little change in Ih. Thus, VIC appears to be produced by a distinct population of HCN channels. This voltage-independent current could contribute significantly to the role of HCN channels in neurons and myocytes; VIC flowing through the channels at physiological potentials would tend to promote excitability by accelerating both depolarization and repolarization.

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Testosterone decreases urinary bladder smooth muscle excitability via novel signaling mechanism involving direct activation of the BK channels

AJP Renal Physiology ◽

10.1152/ajprenal.00238.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. F1253-F1259 ◽

Cited By ~ 8

Author(s):

Kiril L. Hristov ◽

Shankar P. Parajuli ◽

Aaron Provence ◽

Georgi V. Petkov

Keyword(s):

Smooth Muscle ◽

Single Channel ◽

Bk Channels ◽

Bk Channel ◽

Bladder Pressure ◽

Resting Membrane Potential ◽

Open Probability ◽

Membrane Hyperpolarization ◽

Whole Cell ◽

Inside Out

In addition to improving sexual function, testosterone has been reported to have beneficial effects in ameliorating lower urinary tract symptoms by increasing bladder capacity and compliance, while decreasing bladder pressure. However, the cellular mechanisms by which testosterone regulates detrusor smooth muscle (DSM) excitability have not been elucidated. Here, we used amphotericin-B perforated whole cell patch-clamp and single channel recordings on inside-out excised membrane patches to investigate the regulatory role of testosterone in guinea pig DSM excitability. Testosterone (100 nM) significantly increased the depolarization-induced whole cell outward currents in DSM cells. The selective pharmacological inhibition of the large-conductance voltage- and Ca2+-activated K+ (BK) channels with paxilline (1 μM) completely abolished this stimulatory effect of testosterone, suggesting a mechanism involving BK channels. At a holding potential of −20 mV, DSM cells exhibited transient BK currents (TBKCs). Testosterone (100 nM) significantly increased TBKC activity in DSM cells. In current-clamp mode, testosterone (100 nM) significantly hyperpolarized the DSM cell resting membrane potential and increased spontaneous transient hyperpolarizations. Testosterone (100 nM) rapidly increased the single BK channel open probability in inside-out excised membrane patches from DSM cells, clearly suggesting a direct BK channel activation via a nongenomic mechanism. Live-cell Ca2+ imaging showed that testosterone (100 nM) caused a decrease in global intracellular Ca2+ concentration, consistent with testosterone-induced membrane hyperpolarization. In conclusion, the data provide compelling mechanistic evidence that under physiological conditions, testosterone at nanomolar concentrations directly activates BK channels in DSM cells, independent from genomic testosterone receptors, and thus regulates DSM excitability.

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Voltage-dependent dynamics of the BK channel cytosolic gating ring are coupled to the membrane-embedded voltage sensor

eLife ◽

10.7554/elife.40664 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 8

Author(s):

Pablo Miranda ◽

Miguel Holmgren ◽

Teresa Giraldez

Keyword(s):

Conformational Changes ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

Cation Binding ◽

Voltage Sensitivity ◽

Open Probability ◽

Calcium Dependent ◽

Transmembrane Voltage ◽

Voltage Dependent

In humans, large conductance voltage- and calcium-dependent potassium (BK) channels are regulated allosterically by transmembrane voltage and intracellular Ca2+. Divalent cation binding sites reside within the gating ring formed by two Regulator of Conductance of Potassium (RCK) domains per subunit. Using patch-clamp fluorometry, we show that Ca2+ binding to the RCK1 domain triggers gating ring rearrangements that depend on transmembrane voltage. Because the gating ring is outside the electric field, this voltage sensitivity must originate from coupling to the voltage-dependent channel opening, the voltage sensor or both. Here we demonstrate that alterations of the voltage sensor, either by mutagenesis or regulation by auxiliary subunits, are paralleled by changes in the voltage dependence of the gating ring movements, whereas modifications of the relative open probability are not. These results strongly suggest that conformational changes of RCK1 domains are specifically coupled to the voltage sensor function during allosteric modulation of BK channels.

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