S4-S5 Linker Research Articles

A novel bungarotoxin binding site-tagged construct reveals MAPK-dependent Kv4.2 trafficking

Kv4.2 voltage-gated K+ channel subunits, the primary source of the somatodendritic A-type K+ current in CA1 pyramidal neurons of the hippocampus, play important roles in regulating dendritic excitability and plasticity. To better study the trafficking and subcellular distribution of Kv4.2, we created and characterized a novel Kv4.2 construct encoding a bungarotoxin binding site in the extracellular S3–S4 linker region of the α-subunit. When expressed, this construct can be visualized in living cells after staining with rhodamine-conjugated bungarotoxin. We validated the utility of this construct by visualizing the spontaneous internalization and insertion of Kv4.2 in HEK 293T cells. We further report that Kv4.2 colocalized with several endosome markers in HEK 293T cells. In addition, Kv4.2 internalization is significantly impaired by mitogen-activated protein kinase (MAPK) inhibitors in transfected primary hippocampal neurons. Therefore, this newly developed BBS-Kv4.2 construct provides a novel and powerful tool for studying surface Kv4.2 channel localization and trafficking.

Calcium Channel Dysfunction in Epilepsy: Gain of CACNA1E.

De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy With Contractures, Macrocephaly, and Dyskinesias Helbig KL, Lauerer RJ, Bahr JC, et al. Am J Hum Genet. 2019;104(3):562. doi:10.1016/j.ajhg.2018.09.006Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on electroencephalogram (EEG), and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high-voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all 4 S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the antiepileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Human mutations highlight an intersubunit cation–π bond that stabilizes the closed but not open or inactivated states of TRPV channels

An adequate response of a living cell to the ever-changing environment requires integration of numerous sensory inputs. In many cases, it can be achieved even at the level of a single receptor molecule. Polymodal transient receptor potential (TRP) channels have been shown to integrate mechanical, chemical, electric, and thermal stimuli. Inappropriate gating can lead to pathologies. Among the >60 known TRP vanilloid subfamily (V) 4 mutations that interfere with bone development are Y602C or R616Q at the S4-S5 linker. A cation-π bond between the conservative residues Y602 and R616 of neighboring subunits appears likely in many homologous channel structures in a closed state. Our experiments with TRPV4 mutants indicate that the resting-closed state remains stable while the bond is substituted by a salt bridge or disulfide bond, whereas disruption of the contact by mutations like Y602C or R616Q produces gain-of-function phenotypes when TRPV4 is heterologously expressed in the Xenopus oocyte or yeast. Our data indicate that the Y602-R616 cation-π interactions link the four S4-S5 linker helices together, forming a girdle backing the closed gate. Analogous cation-π bonds and the girdle are seen in many closed TRP channel structures. This girdle is not observed in the cryo-EM structure of amphibian TRPV4 (Protein Data Bank ID code 6BBJ), which appears to be in a different impermeable state-we hypothesize this is the inactivated state.

Voltage-dependent activation in EAG channels follows a ligand-receptor rather than a mechanical-lever mechanism

Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, KV10.1 and human ether-a-go-go-related gene (hERG or KV11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5L) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6T) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5L to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in KV10.2 channels. Using cysteine bridges and S4-S5L-mimicking peptides, we show that the ligand/receptor model is conserved in KV10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in KV10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.

Activation of TRPV1 by Lipids: Can Lipid Tails Bridge the Gap between the Vanilloid Binding Site and the Peripheral Cavities?

The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective cation channel, member of the transient receptor potential superfamily of ion channels. TRPV1 is expressed in peripheral sensory neurons, where it transduces noxious stimuli into electrical signals, and is responsible for body temperature regulation, heat sensation, noxious and chronic pain. TRPV1 is activated by a variety of stimuli, including ligand binding at the vanilloid site. We previously discovered that rotation of a conserved residue on the S6 segment from the S4-S5 linker towards the pore is also implicated in TRPV1 activation. This movement is correlated with dehydration of peripheral cavities (PCs) located between S6 and the S4-S5 linker, and with hydration of the pore. However, the structural details for this mechanism remain to be elucidated. Here we explore the hypothesis that annular lipids mediate the allosteric coupling between the vanilloid site and the PCs, ultimately leading to TRPV1 activation. Our multi-microseconds molecular dynamics simulations and free energy calculations of several TRPV1 systems (i.e. TRPV1 bound to vanilloid ligands and/or to the spider venom toxin, Dk/Tx) revealed that annular lipids have a peculiar “buried” conformation upon concomitant binding of vanilloids and Dk/Tx. Interestingly, lipids in this conformation project the hydrophobic tails into the PCs, thereby triggering their dehydration and the consequent opening of the channel. In conclusion, our results elucidate the molecular underpinnings of TRPV1 allosteric regulation by annular lipids, and sheds light on the mechanism by which the binding of vanilloid ligands is coupled to gating motion.

The Conformational Wave in Capsaicin Activation of Transient Receptor Potential Vanilloid 1 Ion Channel

The capsaicin receptor TRPV1 has been intensively studied by cryo-electron microscopy and functional tests. However, though the apo and capsaicin-bound structural models are available, the dynamic process of capsaicin activation remains intangible, largely due to the lack of a capsaicin-induced open structural model and the low occupancy of the transition states. Here we report that reducing temperature toward the freezing point substantially increased channel closure events even in the presence of saturating capsaicin. We further used a combination of fluorescent unnatural amino acid (fUAA) incorporation, computational modeling, and rate-equilibrium linear free-energy relationships analysis (Phi-analysis) to derive the fully open capsaicin-bound state model, and reveal how the channel transits from the apo to the open state. We observed that capsaicin initiates a conformational wave that propagates through the S4-S5 linker towards the S6 bundle and finally reaching the selectivity filter. Our study provides a temporal mechanism for capsaicin activation of TRPV1.

Conserved Residues at the Interface between the S4 and S5 Segments are Critical for Normal Gating of HCN Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have the unique characteristic of being activated at negative membrane potentials, but the mechanism of HCN reverse voltage-dependence is not known. HCN channels share a similar tetrameric architecture with depolarization-activated Kv channels, having six transmembrane domains, where S1-S4 form the voltage-sensing domain (VSD) and S5-S6 form the pore domain (PD). As opposed to Kv channels, HCN channels have a non-domain swapped architecture with a short S4-S5 linker that covalently binds the VSD to the PD. The non-domain swapped architecture implies that the S4 from one subunit directly interacts with the S5 from the same subunit. As well as in depolarization-activated channels, S4 is the positively charged voltage sensor of HCN channels and moves across the membrane in response to changes in the membrane potential. It has been shown before that cutting the S4-S5 linker of HCN channels and other non-domain swapped channels does not impair their typical gating. Therefore, the coupling between the VSD and the PD in HCN channels may involve residues located at the interface between the S4 and S5 segments. The interface between S4 and S5 of HCN channels possesses several residues that are absolutely conserved in the HCN family and that therefore, are potentially important for their unique gating. We individually mutated several of these conserved residues at the S4-S5 interface in the sea urchin HCN (spHCN) channel and expressed them in Xenopus laevis oocytes to measure the effects of each mutation using Voltage Clamp Fluorometry. Our results show that some residues at the S4-S5 interface are critical for normal S4 movement, while others are important to stabilize the gate or are involved in the coupling between S4 and the gate.

Conformational Ensemble of the Human TRPV3 Ion Channel

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand gating.

Continuum Gating Current Models Computed with Consistent Interactions

The action potential of nerve and muscle is produced by voltage-sensitive channels that include a specialized device to sense voltage. The voltage sensor depends on the movement of charges in the changing electric field as suggested by Hodgkin and Huxley. Gating currents of the voltage sensor are now known to depend on the movements of positively charged arginines through the hydrophobic plug of a voltage sensor domain. Transient movements of these permanently charged arginines, caused by the change of transmembrane potential V, further drag the S4 segment and induce opening/closing of the ion conduction pore by moving the S4-S5 linker. This moving permanent charge induces capacitive current flow everywhere. Everything interacts with everything else in the voltage sensor and protein, and so it must also happen in its mathematical model. A Poisson-Nernst-Planck (PNP)-steric model of arginines and a mechanical model for the S4 segment are combined using energy variational methods in which all densities and movements of charge satisfy conservation laws, which are expressed as partial differential equations in space and time. The model computes gating current flowing in the baths produced by arginines moving in the voltage sensor. The model also captures the capacitive pile up of ions in the vestibules that link the bulk solution to the hydrophobic plug. Our model reproduces the signature properties of gating current: 1) equality of ON and OFF charge Q in integrals of gating current, 2) saturating voltage dependence in the Q(charge)-voltage curve, and 3) many (but not all) details of the shape of gating current as a function of voltage. Our results agree qualitatively with experiments and can be improved by adding more details of the structure and its correlated movements. The proposed continuum model is a promising tool to explore the dynamics and mechanism of the voltage sensor.

Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels by interacting with four cytoplasmic channel domains

Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.

Conformational ensemble of the human TRPV3 ion channel

Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating.

De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

A hypothetical molecular mechanism for TRPV1 activation that invokes rotation of an S6 asparagine.

The transient receptor potential channel vanilloid type 1 (TRPV1) is activated by a variety of endogenous and exogenous stimuli and is involved in nociception and body temperature regulation. Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes that occur in the pore domain and result in ionic conduction have not yet been identified. Here we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves rotation of a conserved asparagine in S6 from a position facing the S4-S5 linker toward the pore. This rotation is associated with hydration of the pore and dehydration of the four peripheral cavities located between each S6 and S4-S5 linker. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, evaluate newly available structures, and reexamine previously reported water accessibility and mutagenesis experiments. These analyses provide several independent lines of evidence to support our hypothesis. Finally, we show that our proposed molecular mechanism is compatible with the prevailing theory that the selectivity filter acts as a secondary gate in TRPV1.

Discovery of a Novel Nav1.7 Inhibitor From Cyriopagopus albostriatus Venom With Potent Analgesic Efficacy

Spider venoms contain a vast array of bioactive peptides targeting ion channels. A large number of peptides have high potency and selectivity toward sodium channels. Nav1.7 contributes to action potential generation and propagation and participates in pain signaling pathway. In this study, we describe the identification of μ-TRTX-Ca2a (Ca2a), a novel 35-residue peptide from the venom of Vietnam spider Cyriopagopus albostriatus (C. albostriatus) that potently inhibits Nav1.7 (IC50 = 98.1 ± 3.3 nM) with high selectivity against skeletal muscle isoform Nav1.4 (IC50 > 10 μM) and cardiac muscle isoform Nav1.5 (IC50 > 10 μM). Ca2a did not significantly alter the voltage-dependent activation or fast inactivation of Nav1.7, but it hyperpolarized the slow inactivation. Site-directed mutagenesis analysis indicated that Ca2a bound with Nav1.7 at the extracellular S3–S4 linker of domain II. Meanwhile, Ca2a dose-dependently attenuated pain behaviors in rodent models of formalin-induced paw licking, hot plate test, and acetic acid-induced writhing. This study indicates that Ca2a is a potential lead molecule for drug development of novel analgesics.

Structural insights on TRPV5 gating by endogenous modulators

TRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure reveals a binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure reveals two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.

Structural basis for PtdInsP2-mediated human TRPML1 regulation

Transient receptor potential mucolipin 1 (TRPML1), a lysosomal channel, maintains the low pH and calcium levels for lysosomal function. Several small molecules modulate TRPML1 activity. ML-SA1, a synthetic agonist, binds to the pore region and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a natural lipid, stimulates channel activity to a lesser extent than ML-SA1; moreover, PtdIns(4,5)P2, another natural lipid, prevents TRPML1-mediated calcium release. Notably, PtdIns(3,5)P2 and ML-SA1 cooperate further increasing calcium efflux. Here we report the structures of human TRPML1 at pH 5.0 with PtdIns(3,5)P2, PtdIns(4,5)P2, or ML-SA1 and PtdIns(3,5)P2, revealing a unique lipid-binding site. PtdIns(3,5)P2 and PtdIns(4,5)P2 bind to the extended helices of S1, S2, and S3. The phosphate group of PtdIns(3,5)P2 induces Y355 to form a π-cation interaction with R403, moving the S4–S5 linker, thus allosterically activating the channel. Our structures and electrophysiological characterizations reveal an allosteric site and provide molecular insight into how lipids regulate TRP channels.

Role of the Interaction Motif in Maintaining the Open Gate of an Open Sodium Channel

Voltage-gated sodium channels undergo transitions between open, closed, and inactivated states, enabling regulation of the translocation of sodium ions across membranes. A recently published crystal structure of the full-length prokaryotic NavMs crystal structure in the activated open conformation has revealed the presence of a novel motif consisting of an extensive network of salt bridges involving residues in the voltage sensor, S4-S5 linker, pore, and C-terminal domains. This motif has been proposed to be responsible for maintaining an open conformation that enables ion translocation through the channel. In this study, we have used long-time molecular dynamics calculations without artificial restraints to demonstrate that the interaction network of full-length NavMs indeed prevents a rapid collapse and closure of the gate, in marked difference to earlier studies of the pore-only construct in which the gate had to be restrained to remain open. Interestingly, a frequently discussed “hydrophobic gating” mechanism at nanoscopic level is also observed in our simulations, in which the discontinuous water wire close to the gate region leads to an energetic barrier for ion conduction. In addition, we demonstrate the effects of in silico mutations of several of the key residues in the motif on the open channel’s stability and functioning, correlating them with existing functional studies on this channel and homologous disease-associated mutations in human sodium channels; we also examine the effects of truncating/removing the voltage sensor and C-terminal domains in maintaining an open gate.

Effects of β-subunit on gating of a potassium ion channel: Molecular simulations of cardiac IKs activation

Dynamic conformational changes of ion channel proteins during activation gating determine their function as carriers of current. The relationship between these molecular movements and channel function over the physiological timescale of the action potential (AP) has not been fully established due to limitations of existing techniques. We constructed a library of possible cardiac IKs protein conformations and applied a combination of protein segmentation and energy linearization to study this relationship computationally. Simulations reproduced the effects of the beta-subunit (KCNE1) on the alpha-subunit (KCNQ1) dynamics and function, observed in experiments. Mechanistically, KCNE1 increased the probability of “visiting” conducting pore conformations on activation trajectories, thereby increasing IKs current. KCNE1 slowed IKs activation by impeding the voltage sensor (VS) movement and reducing its coupling to pore opening. Conformational changes along activation trajectories determined that the S4-S5 linker (S4S5L) plays an important role in these modulatory effects by KCNE1. Integration of these molecular structure-based IKs dynamics into a model of human cardiac ventricular myocyte, revealed that KCNQ1-KCNE1 interaction is essential for normal AP repolarization.

The N terminus and transmembrane segment S1 of Kv1.5 can coassemble with the rest of the channel independently of the S1–S2 linkage

The voltage-gated potassium channel Kv1.5 belongs to the Shaker superfamily. Kv1.5 is composed of four subunits, each comprising 613 amino acids, which make up the N terminus, six transmembrane segments (S1-S6), and the C terminus. We recently demonstrated that, in HEK cells, extracellularly applied proteinase K (PK) cleaves Kv1.5 channels at a single site in the S1-S2 linker. This cleavage separates Kv1.5 into an N-fragment (N terminus to S1) and a C-fragment (S2 to C terminus). Interestingly, the cleavage does not impair channel function. Here, we investigated the role of the N terminus and S1 in Kv1.5 expression and function by creating plasmids encoding various fragments, including those that mimic PK-cleaved products. Our results disclosed that although expression of the pore-containing fragment (Frag(304-613)) alone could not produce current, coexpression with Frag(1-303) generated a functional channel. Immunofluorescence and biotinylation analyses uncovered that Frag(1-303) was required for Frag(304-613) to traffic to the plasma membrane. Biochemical analysis revealed that the two fragments interacted throughout channel trafficking and maturation. In Frag(1-303)+(304-613)-coassembled channels, which lack a covalent linkage between S1 and S2, amino acid residues 1-209 were important for association with Frag(304-613), and residues 210-303 were necessary for mediating trafficking of coassembled channels to the plasma membrane. We conclude that the N terminus and S1 of Kv1.5 can attract and coassemble with the rest of the channel (i.e. Frag(304-613)) to form a functional channel independently of the S1-S2 linkage.

Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are both voltage- and ligand-activated membrane proteins that contribute to electrical excitability and pace-making activity in cardiac and neuronal cells. These channels are members of the voltage-gated Kv channel superfamily and cyclic nucleotide-binding domain subfamily of ion channels. HCN channels have a unique feature that distinguishes them from other voltage-gated channels: the HCN channel pore opens in response to hyperpolarizing voltages instead of depolarizing voltages. In the canonical model of electromechanical coupling, based on Kv channels, a change in membrane voltage activates the voltage-sensing domains (VSD) and the activation energy passes to the pore domain (PD) through a covalent linker that connects the VSD to the PD. In this investigation, the covalent linkage between the VSD and PD, the S4-S5 linker, and nearby regions of spHCN channels were mutated to determine the functional role each plays in hyperpolarization-dependent activation. The results show that: (i) the S4-S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating; (ii) the S4 C-terminal region (S4C-term) is not necessary for ligand-dependent gating but is required for hyperpolarization-dependent activation and acts like an autoinhibitory domain on the PD; (iii) the S5N-term region is involved in VSD-PD coupling and holding the pore closed; and (iv) spHCN channels have two voltage-dependent processes, a hyperpolarization-dependent activation and a depolarization-dependent recovery from inactivation. These results are inconsistent with the canonical model of VSD-PD coupling in Kv channels and elucidate the mechanism for hyperpolarization-dependent activation of HCN channels.