Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein–protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP₂). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP₂. This result is due to loss of coupling because PIP₂ was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP₂-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K⁺ channels, suggesting that lipids play an important role in coupling in many ion channels.     

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane

Voltage-dependent K⁺ (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K⁺ channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane.     

Cadmium–cysteine coordination in the BK inner pore region and its structural and functional implications

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd²⁺ coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd²⁺ forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K⁺ (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd²⁺ binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltage-dependent gating between these channels.How transmembrane potential influences the opening and closing of ion channels, a process known as gating, is central to understanding how cellular excitability is regulated (1). For voltage-dependent K⁺ (Kv) channels, functional (2–4) and crystallographic (5, 6) studies have led to a compelling model of gating. In this model there are two key elements, both of which arise from properties of the cytosolic end of Kv channels: a cytosolic ion permeation gate formed by an interlaced arrangement of S6 inner helices termed the bundle crossing (2, 3), and a kink produced by the conserved proline-valine-proline (PVP) motif (Fig. 1A, boxed residues) to allow the C terminus of Kv S6 to form extensive contact with the S4–S5 linker (5, 6). Thus, the outward movement of the voltage sensors (VSDs) induced by transmembrane depolarization is thought to be transmitted to S6 through the S4–S5 linker to open the cytosolic ion permeation gate (6–8), and this enables access of cytosolic K⁺ ions to the Kv inner pore region.Open in a separate windowFig. 1.Cd²⁺ coordinates with cysteines separately introduced at two BK inner pore sites. (A) Sequence alignment of the S6 segment in the inner pore regions of Slo family channels, Kv channels, and the KcsA channel. The conserved glycine hinge and prolines in the PVP motif (boxed residues) are colored in red. The sites evaluated with cysteine substitution in BK S6 are underlined. A316 and V319 are indicated by arrowheads. (B) Radius profiles of Kv1.2 (blue) and KcsA (red) channels calculated from their crystal structures. The approximate locations of A316C and V319C are marked by horizontal dotted lines. SF, selectivity filter; IP, inner pore; BC, bundle crossing. (C–E) Currents of WT, V319C, and A316C recorded in 300 μM Ca²⁺ without (Left) and with (Right) 100 μM intracellular Cd²⁺. Voltage protocol was shown in E. (F) G–V relationships of WT (black circles), V319C (red triangles), and A316C (blue squares) in 300 μM Ca²⁺ without (open symbols) or with (filled symbols) 100 μM Cd²⁺. Boltzmann fit results (lines) are z = 1e, V_h = −3 mV (WT, control); z = 0.75e, V_h = −57 mV (WT, Cd²⁺); z = 0.94e, V_h = +69 mV (V319C, control); z = 0.6e, V_h = +136 mV (A316C, control); z = 0.32e, V_h = −66 mV (A316C, Cd²⁺).Although this model is generally accepted for Kv channel gating, it is not clear to what extent it applies to other K⁺ channels. For example, the large conductance, Ca²⁺-activated K⁺ (BK or Slo1) channel shares with Kv channels a similar set of four VSDs attached to a central pore and gate domain (PGD), such that both channels are voltage dependent (9). However, unlike that of Kv channels, the inner pore of a closed BK channels is accessible to large molecules such as quaternary ammonium (QA) blockers (10, 11) and methanethiosulfonate ethyltrimethylammonium (MTSET) (12), indicating that the cytosolic end of a closed BK channel cannot completely occlude K⁺ flow; this indicates that a gate extracellular to that proposed for Kv channels is required to securely prevent K⁺ flow in a closed BK channel. As a corollary, the underlying structural and conformational details required to couple VSD activation to channel opening may differ between BK and Kv channels.To provide new insight into differences between BK and Kv in the inner pore region, we have probed the cadmium (Cd²⁺) sensitivity of cysteine residues introduced in the BK S6 inner helix. Compared with cysteine modification by MTSET (12), Cd²⁺–Cys coordination provides two potential advantages for investigation of BK channel gating. First, Cd²⁺ coordination can involve multiple residues with strict distance and geometric requirements. This potentially provides information about the relative position of coordinating residues in the BK inner pore region, thereby placing important structural constraints on BK S6. Second, the size of Cd²⁺ is closer to that of a K⁺ ion compared with large probes such as QA blockers or MTSET; this allows a more accurate evaluation of whether there may be restriction to K⁺ flow at the cytosolic end of BK channel. Despite the evidence that large molecules can access the BK inner cavity in closed states, gated access of intracellularly applied Shaker ball peptide indicates that conformational changes do occur at the cytosolic end of BK channels (13). Furthermore, our previous study showed that the open-state modification of BK inner pore cysteines by MTSET is approximately two orders of magnitude faster than that in closed states (12), although the state dependence is weaker than that observed in Shaker channels (3). It is therefore important to determine the extent to which the BK cytosolic side may restrict inner pore access of K⁺ by using a probe similar in size to K⁺.Here we focus on two BK S6 residues, V319 and A316, which confer interesting sensitivity to Cd²⁺ on BK channels when replaced by cysteine. V319 is in register with Kv residues that contribute to the bundle crossing in Kv channels. The results reveal that Cd²⁺ coordination occurs between V319C and E321 in the same BK α-subunit, requiring that BK S6 adopts a novel kink at this level that is distinct from that in Kv channel structures. Furthermore, for A316C, which lines the BK inner pore, the Cd²⁺ coordination rate differs only modestly between open and closed states, suggesting there is no significant restriction of K⁺ access to the BK inner cavity in both states.     

Voltage-sensor movements in the Eag Kv channel under an applied electric field

Voltage-dependent ion channels regulate the opening of their pores by sensing the membrane voltage. This process underlies the propagation of action potentials and other forms of electrical activity in cells. The voltage dependence of these channels is governed by the transmembrane displacement of the positive charged S4 helix within their voltage-sensor domains. We use cryo-electron microscopy to visualize this movement in the mammalian Eag voltage-dependent potassium channel in lipid membrane vesicles with a voltage difference across the membrane. Multiple structural configurations show that the applied electric field displaces S4 toward the cytoplasm by two helical turns, resulting in an extended interfacial helix near the inner membrane leaflet. The position of S4 in this down conformation is sterically incompatible with an open pore, thus explaining how movement of the voltage sensor at hyperpolarizing membrane voltages locks the pore shut in this kind of voltage-dependent K⁺ (K_v) channel. The structures solved in lipid bilayer vesicles detail the intricate interplay between K_v channels and membranes, from showing how arginines are stabilized deep within the membrane and near phospholipid headgroups, to demonstrating how the channel reshapes the inner leaflet of the membrane itself.

In voltage-dependent ion channels, the transmembrane voltage determines whether the pore opens. At the same time, the flow of ions through the open pore alters the membrane voltage by charging the membrane capacitance. This recursive regulation of ion channel activity by membrane voltage is the fundamental process at the heart of cellular electricity. As described by Hodgkin and Huxley (1), voltage-dependent membrane permeability to Na⁺ and K⁺ (ion channels as molecular entities had not yet been discovered) generates the action potential, which is by far the most rapid form of information transfer across long distances in cells. Voltage-dependent ion channels underlie many other aspects of cell signaling as well, including the initiation of muscle contraction by voltage-dependent Ca²⁺ channels (2, 3) and the control of cardiac and neuronal pacemaker frequency by the hyperpolarization-activated cyclic nucleotide–gated (HCN) channel (4, 5).Voltage-dependent ion channels contain structural domains called voltage sensors that control pore opening by membrane voltage. Whether in K⁺, Na⁺, Ca²⁺, or cation channels like HCN channels or transient receptor potential (TRP) channels, voltage sensors have a conserved structure comprising four transmembrane helices, named S1, S2, S3, and S4 (3, 6, 7). The fourth helix, S4, contains repeats of the amino acid triplet (RXX)_n, where R stands for arginine, sometimes substituted by lysine, X for hydrophobic amino acid, and n varies widely among different channels. At the center of voltage sensors, inside the membrane’s interior, a constellation of negative charged amino acids, aspartate or glutamate, and a phenylalanine residue forms a gating-charge transfer center that stabilizes the positive charged side chains of arginine and lysine as they cross the membrane (8, 9). In some voltage sensors, S4 undergoes a transition from an α to a 3₁₀ helix to direct the arginine and lysine side chains of S4 into the gating-charge transfer center (8–11). The displacement of S4 across the membrane is detectable as a nonlinear capacitive current, called gating current in electrophysiology experiments (9, 12), and is ultimately responsible for voltage control of a voltage-dependent ion channel’s pore.So far, voltage-dependent ion channel structures have been determined in crystals, detergent micelles, or nanodiscs without a voltage difference across them (8, 13–19). Under such conditions, most voltage sensors adopt a depolarized conformation, which is expected in a membrane at 0 mV. Chemical cross links, metal affinity bridges, mutations, and toxins have been used to capture or stabilize voltage sensors in conformations thought to mimic the hyperpolarized (i.e., negative voltage inside) condition (10, 11, 20–23). Here, we present a cryo-electron microscopy (cryo-EM) analysis of the mammalian Eag voltage-dependent K⁺ (K_v) channel in lipid membrane vesicles with a voltage generated across the membrane using K⁺ ion gradients in the presence of valinomycin. Doing so allows us to not only visualize how the voltage sensors respond to the applied electric field but also to see how the lipid membrane near the channel, which is intimately tied to the function of voltage-dependent ion channels, is reshaped by these conformational changes.     

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K⁺) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K⁺ channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K⁺ channel inactivation and exhibits a remarkable structural plasticity that correlates to K⁺ channel inactivation. The transmembrane helix 1 unwinds when the K⁺ channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K⁺ ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K⁺ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K⁺ channel pore.     

Localization-dependent activity of the Kv2.1 delayed-rectifier K+ channel

The Kv2.1 K⁺ channel is highly expressed throughout the brain, where it regulates excitability during periods of high-frequency stimulation. Kv2.1 is unique among Kv channels in that it targets to large surface clusters on the neuronal soma and proximal dendrites. These clusters also form in transfected HEK cells. Following excessive excitatory stimulation, Kv2.1 declusters with an accompanying 20- to 30-mV hyperpolarizing shift in the activation threshold. Although most Kv2.1 channels are clustered, there is a pool of Kv2.1 resident outside of these domains. Using the cell-attached patch clamp technique, we investigated the hypothesis that Kv2.1 activity varies as a function of cell surface location. We found that clustered Kv2.1 channels do not efficiently conduct K⁺, whereas the nonclustered channels are responsible for the high threshold delayed rectifier K⁺ current typical of Kv2.1. Comparison of gating and ionic currents indicates only 2% of the surface channels conduct, suggesting that the clustered channels still respond to membrane potential changes. Declustering induced via either actin depolymerization or alkaline phosphatase treatment did not increase whole-cell currents. Dephosphorylation resulted in a 25-mV hyperpolarizing shift, whereas actin depolymerization did not alter the activation midpoint. Taken together, these data demonstrate that clusters do not contain high threshold Kv2.1 channels whose voltage sensitivity shifts upon declustering; nor are they a reservoir of nonconducting channels that are activated upon release. On the basis of these findings, we propose unique roles for the clustered Kv2.1 that are independent of K⁺ conductance.     

Expression of voltage-gated K<Superscript>+</Superscript> channels in human atrium

Voltage-gated K⁺ channels underlie repolarisation of the cardiac action potential and represent a potential therapeutic target in the treatment of cardiac dysrhythmias. However, very little is known about the relative expression of K⁺ channel subunits in the human myocardium. We used a semi-quantitative RT-PCR technique to examine the relative expression of mRNAs for the voltage-gated K⁺ channel subunits, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, KvLQT1, HERG and IsK in samples of human atrial appendage. Data were expressed as a percentage expression density relative to an 18S ribosomal RNA internal standard. The most abundant K⁺ channel mRNAs were Kv4.3 (80.7 ± 10.1 %), Kv1.5 (69.7 ± 11.2 %) and HERG (55.9 ± 21.5 %). Significant expression of KvLQT1 (33.5 ± 5.5 %,) and Kv1.4 (26.7 ± 9.6 %) was also detected. Levels of mRNAs for Kv1.2 and IsK were very low and neither Kv2.1 nor Kv4.2 mRNA were detected in any experiments. Whole-cell patch-clamp techniques were used to examine the outward currents of isolated human atrial myocytes at 37 °C. These recordings demonstrated the existence of transient (I_to1) and sustained (I_so) outward currents in isolated human atrial myocytes. I_to1, and not I_so, showed voltage-dependent inactivation during 100 ms pre-pulses. Both I_to1 and I_so were inhibited by high concentrations (2 mM) of the K⁺ channel blocker, 4-aminopyridine (4-AP). However, lower concentrations of 4-AP (10 μM) inhibited I_so selectively. I_to1 recovered from inactivation relatively rapidly (t ∼21 ms). These data, with published information regarding the properties of expressed K⁺ channels, suggest that Kv4.3 represents the predominant K⁺ channel subunit underlying I_to1 with little contribution of Kv1.4. The sensitivity of Iso to very low concentrations of 4-aminopyridine and the relatively low expression of mRNA for Kv1.2 and Kv2.1 is consistent with the major contribution of Kv1.5 to this current. The physiological significance of the expression of KvLQT1 and Kv1.4 mRNA in the human atrium warrants further investigation. Received: 30 August 2000, Returned for 1. revision: 21 September 2000, 1. Revision received: 21 June 2002, Returned for 2. revision: 15 July 2002, 2. Revision received: 30 July 2002, Accepted: 31 July 2002 Correspondence to: Dr. A. F. James     

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

Mutations in the K_V7.2 gene encoding for voltage-dependent K⁺ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S₄ domain of K_V7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K_V7.2 and/or K_V7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying K_V7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal K_v7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K⁺ channel impairment, and set the preclinical basis for the potential use of K_v7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K_V7.2 encephalopathy.     

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide–Kv1.3). Both the apo-Kv1.3 and dalazatide–Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K⁺ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide–Kv1.3, binding of dalazatide to the channel’s outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K⁺ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3’s transition into the drug-blocked state.

Potassium channels form K⁺-selective pores that span cell membranes in virtually all living organisms. In humans, a family of 78 genes encodes four classes of K⁺ channels (voltage-gated, calcium-activated, inward rectifier, and two-pore channels), which are involved in a multitude of physiological functions in both electrically excitable and nonexcitable cells (1). All four classes of channels conduct K⁺ ions selectively and rapidly, but they differ in how they are gated. The selectivity filter is the structural element responsible for the exquisitely K⁺-selective pore (2–5). It is the narrowest part of the ion conduction pathway and connects a water-filled cavity in the center of the protein with an outer vestibule in the extracellular solution. The filter accommodates K⁺ ions at four sites called S1, S2, S3, and S4 starting at the extracellular side. The signature sequence G(Y/F)G in the selectivity filter plays a critical role in making the pore K⁺ selective (6, 7). In all K⁺ channel structures determined, both bacterial and eukaryotic, the aromatic residue (Y or F) in the signature sequence is nearly identical in position, although these channels differ in the conformation (closed or open) of the S6 helical inner gate (8). In the hERG/Kv11.1 channel, a subtle deviation in the position of F627 in the signature sequence causes a slight widening of the selectivity filter, which has been suggested to underlie the channel’s transition into the C-type inactivated state (8).The voltage-gated potassium channel (Kv) Kv1.3–Kvβ2 in lymphocytes and microglia provides the counterbalancing cation efflux to promote calcium entry necessary for calcium signaling (9, 10). Selective blockers of Kv1.3–Kvβ2 treat diverse autoimmune and neuroinflammatory diseases in rodent models (9, 10), highlighting the channel’s physiological and pharmacological importance. Here, we determined structures of Kv1.3 complexed to its accessory subunit Kvβ2 alone (apo-Kv1.3) and bound to dalazatide (dalazatide–Kv1.3), a potent and selective peptide inhibitor of Kv1.3 in clinical trials for autoimmune and neuroinflammatory diseases (10–14). Both apo-Kv1.3 and dalazatide–Kv1.3 are in the activated state based on the depolarized voltage sensor and open S6 helical inner gate. Comparison of the two structures reveals substantial conformational changes in the selectivity filter. In apo-Kv1.3, Y447 in the signature sequence diverges more than 11 Å from the position of corresponding aromatic residues in other K⁺ channels, both in eukaryotes and bacteria. The outer pore is wider at S1 and S2 and narrowed at S0 K⁺-binding sites, resulting in loss of the K⁺ ion from site S2. A network of intrasubunit hydrogen bonds (H451–Y447, H451–D449) stabilizes this unique conformation of the selectivity filter of apo-Kv1.3, and, interestingly, the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is absent. Apo-Kv1.3’s selectivity filter and voltage-sensing domain (VSD) differ significantly from two structures of Kv1.3 that were recently described (16). In dalazatide–Kv1.3, dalazatide’s interaction with H451 disrupts the H451–Y447 hydrogen bond, freeing Y447 to swing back into the interior of the selectivity filter and adopt a position seen in other K⁺ channels. The selectivity filter is narrower, and K⁺ ions are present at sites S2–S4 but not at S1. This conformation is stabilized by a network of intersubunit hydrogen bonds (Y447–W437, Y447–T441, and H451–D449), but the intrasubunit hydrogen bond (W436–D449) that prevents C-type inactivation (15) is likely absent. Our structures provide a basis for the design of Kv1.3 inhibitors for use as immunomodulatory therapeutics.     

Toward a Molecular Understanding of Voltage-Gated Potassium Channels

Voltage-Gated Potassium Channels . Many different types of potassium (K⁺) channels exist and they play a central role in the fine tuning of excitability properties. Of the distinct subpopulations of K⁺ channels expressed in different cells, voltage-gated K⁺ channels have been studied most thoroughly at a molecular level. Over the last few years, the joint application of recombinant DNA technology together with electrophysiology, such as the voltage clamp and the patch clamp techniques, has produced a wealth of information. We have begun to unravel the genetic basis of ion channel diversity. In particular, the Xenopus oocyte expression system has turned out to be of enormous experimental value. Oocytes microinjected with “cloned” mRNA have been used to gain insight into biophysical and pharmacologic properties of voltage-gated K⁺, Na⁺, and Ca²⁺ channels. Here, we will review our understanding of K⁺ channel diversity based upon the fact that ion channels are encoded as a large multigene family. We have caught a first glimpse at possible molecular mechanisms underlying several biophysical properties characteristic for voltage-gated ion channels: voltage dependence of activation and inactivation, and ion permeation and selectivity. We will discuss molecular mechanisms of K⁺ channel activation and inactivation. We will also describe experiments that led to the identification of the “pore region,” and we will present a model of a potassium selective ion channel pore.     

Modulation of Kv Channel α/β Subunit Interactions

Voltage-gated K⁺ channels comprise the largest and most diverse class of ion channels. These channels establish the resting membrane potential and modulate the frequency and duration of action potentials in nerve and muscle, as well as being the targets of several antiarrhythmic drugs in the heart. The multiplicity of Kv channel function is further enhanced through modulation by accessory β subunits, which confer rapid inactivation, alter current amplitudes, and promote cell surface expression. In addition, α/β interactions are also influenced by second messenger pathways. Recent evidence demonstrates that phosphorylation of Kv channel α and/or β subunits may dramatically affect channel properties. The functional response of different K⁺ channel subunits to activation of protein kinases represents not only a means to modulate subunit interactions, but also another mechanism for K⁺ channel diversity in vivo.     

Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1–S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca²⁺-activated K⁺ channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its “decentralized” distribution of voltage-sensing residues, we probed the BK_Ca VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.     

Autism-associated mutations in KV7 channels induce gating pore current

Autism spectrum disorder (ASD) adversely impacts >1% of children in the United States, causing social interaction deficits, repetitive behaviors, and communication disorders. Genetic analysis of ASD has advanced dramatically through genome sequencing, which has identified >500 genes with mutations in ASD. Mutations that alter arginine gating charges in the voltage sensor of the voltage-gated potassium (K_V) channel K_V7 (KCNQ) are among those frequently associated with ASD. We hypothesized that these gating charge mutations would induce gating pore current (also termed ω-current) by causing an ionic leak through the mutant voltage sensor. Unexpectedly, we found that wild-type K_V7 conducts outward gating pore current through its native voltage sensor at positive membrane potentials, owing to a glutamine in the third gating charge position. In bacterial and human K_V7 channels, gating charge mutations at the R1 and R2 positions cause inward gating pore current through the resting voltage sensor at negative membrane potentials, whereas mutation at R4 causes outward gating pore current through the activated voltage sensor at positive potentials. Remarkably, expression of the K_V7.3/R2C ASD-associated mutation in vivo in midbrain dopamine neurons of mice disrupts action potential generation and repetitive firing. Overall, our results reveal native and mutant gating pore current in K_V7 channels and implicate altered control of action potential generation by gating pore current through mutant K_V7 channels as a potential pathogenic mechanism in autism.

Autism spectrum disorder (ASD) is a neurological developmental disability that generally appears in children before the age of 3 y (1). ASD results in a wide spectrum of symptoms, including deficits in social interaction, cognitive function, and communication skills and repetitive behaviors. ASD is caused by a complex interaction of environmental and genetic factors. Recently, our understanding of the genetic causes of ASD has increased significantly. There is a high genetic heritability in autism, and complete genome sequencing of an affected individual plus an unaffected sibling and two unaffected parents (trios) has identified more than 500 genes with mutations that are now linked to ASD (2, 3). Cluster analysis of these genes revealed an unexpected number of voltage-gated ion channels (4), and further sequence comparisons revealed overrepresentation of mutations in the Arg residues that serve as the gating charges of their voltage sensors (SI Appendix, Fig. S1) (4). Such mutations are expected to alter the voltage-dependent control of opening and closing of ion channels.The voltage-gated potassium (K_V) channel K_V7 (KCNQ) ranked high among the ion channel genes having mutations in individuals with neurodevelopmental disorders (4). The K_V7 channels in brain are formed from heterotetramers of K_V7.2 and K_V7.3, K_V7.3 and K_V7.4, or K_V7.3 and K_V7.5 (5), with K_V7.2/7.3 being the most prevalent (Fig. 1A) (6, 7). K_V7 heteromultimers conduct a tonic inhibitory current that is modulated by Gq-coupled receptors, most notably muscarinic acetylcholine receptors, for which K_V7 current is often termed M current (5, 6, 8). K_V7 heteromultimers are expressed in both excitatory and inhibitory neurons, particularly at axon initial segments and at nodes of Ranvier (9), where they help to set the resting membrane potential and regulate the firing threshold and frequency. Conditional deletion of K_V7.2 from cerebral cortex causes hyperexcitability (10). Deletion of K_V7.2/7.3 channels also causes hyperexcitability of inhibitory parvalbumin-expressing interneurons, but this ultimately leads to homeostatic potentiation of excitatory transmission between pyramidal neurons (11). Genetic or pharmacological reduction of the K_V7 current causes epilepsy (e.g., benign familial neonatal convulsions) (12, 13). As these examples demonstrate, K_V7 channels are broadly important in control of action potential firing and in loss of control of electrical excitability in neurological disease, and as such, pharmacological interventions at K_V7 channels may have value in multiple neurological and psychiatric diseases (14).Open in a separate windowFig. 1.Gating pore currents in bacterial potassium channel K_V7Oc. (A) Bioinformatic analysis of ASD mutations in ion channels. (B) Central pore K⁺ currents (Inset) and conductance–voltage (G–V) curve for K_V7Oc during 200-ms depolarizations from −100 mV to the indicated potentials. (C) Wild-type gating pore Cs⁺ currents (I_gp) (Inset) from different holding potentials: −100 mV (black squares), −50 mV (gray squares), and 0 mV (open squares). n = 5 to 10. (D) Blocking of wild-type Cs⁺gating pore current by Q3R mutation. P = 0.001; Student’s t test, two sided.Like other K_V channels, K_V7 is composed of four subunits having six transmembrane segments (S1 to S6) (15). The S1 to S4 segments form the voltage sensor, whereas the S5 and S6 segments form the pore (15). The S4 segments of K_V channels generally contain four or more repeated motifs of a positively charged residue, usually arginine (Arg), flanked by two hydrophobic residues (15). These positively charged residues serve as gating charges that respond to depolarization and repolarization by moving outward and inward across the hydrophobic constriction site, which seals the voltage sensor (15–17). Voltage-dependent movements of the S4 segment driven by the gating charges are conformationally coupled to opening and closing the pore (15–17).The gating charges of voltage-gated sodium and calcium channels in skeletal muscle are targets of mutations that cause hypokalemic periodic paralysis (18). Remarkably, mutation of the gating charge Arg residues causes an ionic leak through the voltage sensor, termed ω-current or gating pore current (19–23). Continuous inward leak of Na⁺ through the mutant voltage sensor reduces the sodium gradient in skeletal muscle fibers, depolarizes and creates instability of the membrane potential, and depresses firing of action potentials, leading to flaccid paralysis (21–23). This suggests that gating charge mutations in K_V7 channels could cause gating pore current that is pathogenic in ASD.We unexpectedly detected robust outward gating pore current in wild-type (WT) K_V7 channels, which depends on a highly conserved Gln (Q) at the third gating charge position that is a conserved Arg (R) in other voltage-gated channels (15). Introducing the ASD-associated gating charge mutations R1Q and R2C at the first and second gating charge positions in K_V7.3 generated inward gating pore current through the mutant voltage sensor, and the ASD-associated gating charge mutation R4C at the fourth gating charge position generated increased outward gating pore current. Dopamine neurons of the ventral tegmental area (VTA) are potently modulated by M current generated by K_V7 channels, which strongly regulates action potential firing and input–output relationships (24–26). These cells play a key role in the regulation of neural networks that control social behavior (27–29), and disrupted function of dopamine neurons is increasingly linked to ASD etiology (30–33). We found that replacing mouse wild-type K_V7.3 with human K_V7.3 containing the ASD mutation R2C markedly disrupted action potential firing in dopamine neurons in brain slices. Collectively, our results support the hypothesis that gating pore current through mutant voltage sensors of K_V7 channels disrupts neural activity and implicate pathogenic gating pore current as an important contributor to ASD.     

Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.     

Calix[4]arene-based conical-shaped ligands for voltage-dependent potassium channels

Potassium channels are among the core functional elements of life because they underpin essential cellular functions including excitability, homeostasis, and secretion. We present here a series of multivalent calix[4]arene ligands that bind to the surface of voltage-dependent potassium channels (K_v1.x) in a reversible manner. Molecular modeling correctly predicts the best candidates with a conical C₄ symmetry for optimal binding, and the effects on channel function are assessed electrophysiologically. Reversible inhibition was observed, without noticeable damage of the oocytes, for tetraacylguanidinium or tetraarginine members of the series with small lower rim O-substituents. Apparent binding constants were in the low micromolar range and had Hill coefficients of 1, consistent with a single site of binding. Suppression of current amplitude was accompanied by a positive shift in the voltage dependence of gating and slowing of both voltage sensor motion and channel opening. These effects are in keeping with expectations for docking in the central pore and interaction with the pore domain “turret.”     

Kv4.3 is not required for the generation of functional Ito,f channels in adult mouse ventricles

Accumulated evidence suggests that the heteromeric assembly of Kv4.2 and Kv4.3 α-subunits underlies the fast transient Kv current (I_to,f) in rodent ventricles. Recent studies, however, demonstrated that the targeted deletion of Kv4.2 results in the complete elimination of I_to,f in adult mouse ventricles, revealing an essential role for the Kv4.2 α-subunit in the generation of mouse ventricular I_to,f channels. The present study was undertaken to investigate directly the functional role of Kv4.3 by examining the effects of the targeted disruption of the KCND3 (Kv4.3) locus. Mice lacking Kv4.3 (Kv4.3−/−) appear indistinguishable from wild-type control animals, and no structural or functional abnormalities were evident in Kv4.3−/− hearts. Voltage-clamp recordings revealed that functional I_to,f channels are expressed in Kv4.3−/− ventricular myocytes, and that mean I_to,f densities are similar to those recorded from wild-type cells. In addition, I_to,f properties (inactivation rates, voltage dependences of inactivation and rates of recovery from inactivation) in Kv4.3−/− and wild-type mouse ventricular myocytes were indistinguishable. Quantitative RT-PCR and Western blot analyses did not reveal any measurable changes in the expression of Kv4.2 or the Kv channel interacting protein (KChIP2) in Kv4.3−/− ventricles. Taken together, the results presented here suggest that, in contrast with Kv4.2, Kv4.3 is not required for the generation of functional mouse ventricular I_to,f channels.     

Filter gate closure inhibits ion but not water transport through potassium channels

The selectivity filter of K⁺ channels is conserved throughout all kingdoms of life. Carbonyl groups of highly conserved amino acids point toward the lumen to act as surrogates for the water molecules of K⁺ hydration. Ion conductivity is abrogated if some of these carbonyl groups flip out of the lumen, which happens (i) in the process of C-type inactivation or (ii) during filter collapse in the absence of K⁺. Here, we show that K⁺ channels remain permeable to water, even after entering such an electrically silent conformation. We reconstituted fluorescently labeled and constitutively open mutants of the bacterial K⁺ channel KcsA into lipid vesicles that were either C-type inactivating or noninactivating. Fluorescence correlation spectroscopy allowed us to count both the number of proteoliposomes and the number of protein-containing micelles after solubilization, providing the number of reconstituted channels per proteoliposome. Quantification of the per-channel increment in proteoliposome water permeability with the aid of stopped-flow experiments yielded a unitary water permeability p_f of (6.9 ± 0.6) × 10⁻¹³ cm³⋅s⁻¹ for both mutants. “Collapse” of the selectivity filter upon K⁺ removal did not alter p_f and was fully reversible, as demonstrated by current measurements through planar bilayers in a K⁺-containing medium to which K⁺-free proteoliposomes were fused. Water flow through KcsA is halved by 200 mM K⁺ in the aqueous solution, which indicates an effective K⁺ dissociation constant in that range for a singly occupied channel. This questions the widely accepted hypothesis that multiple K⁺ ions in the selectivity filter act to mutually destabilize binding.     

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.     

Molecular determinants of cardiac transient outward potassium current (Ito) expression and regulation

Rapidly activating and inactivating cardiac transient outward K⁺ currents, I_to, are expressed in most mammalian cardiomyocytes, and contribute importantly to the early phase of action potential repolarization and to plateau potentials. The rapidly recovering (I_to,f) and slowly recovering (I_to,s) components are differentially expressed in the myocardium, contributing to regional heterogeneities in action potential waveforms. Consistent with the marked differences in biophysical properties, distinct pore-forming (α) subunits underlie the two I_to components: Kv4.3/Kv4.2 subunits encode I_to,f, whereas Kv1.4 encodes I_to,s, channels. It has also become increasingly clear that cardiac I_to channels function as components of macromolecular protein complexes, comprising (four) Kvα subunits and a variety of accessory subunits and regulatory proteins that influence channel expression, biophysical properties and interactions with the actin cytoskeleton, and contribute to the generation of normal cardiac rhythms. Derangements in the expression or the regulation of I_to channels in inherited or acquired cardiac diseases would be expected to increase the risk of potentially life-threatening cardiac arrhythmias. Indeed, a recently identified Brugada syndrome mutation in KCNE3 (MiRP2) has been suggested to result in increased I_to,f densities. Continued focus in this area seems certain to provide new and fundamentally important insights into the molecular determinants of functional I_to channels and into the molecular mechanisms involved in the dynamic regulation of I_to channel functioning in the normal and diseased myocardium.     

Family of prokaryote cyclic nucleotide-modulated ion channels

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24–32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K⁺ ions over Na⁺ ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels belong to the superfamily of voltage-gated K⁺ channels. Both types of channels share a similar domain topology with six transmembrane domains, a C-linker region, and a cyclic nucleotide binding domain (CNBD). The S5–S6 segment forms the channel pore, including the selectivity filter for cations. The S4 segment contains several positively charged amino acids, suggesting that it acts as voltage sensor. Despite these similarities in sequence, the function of HCN and CNG channels is noticeably different: HCN channels activate upon membrane hyperpolarization and can be modulated by cyclic nucleotides. They are weakly selective for K⁺ over Na⁺ ions (for reviews, see refs. 1–3). In contrast, CNG channels are activated by the binding of cyclic nucleotides solely and their activity depends only weakly on voltage. The ionic current is carried by both monovalent and divalent cations (for reviews, see refs. 4 and 5).Insight into the structure of HCN channels has been gained only from crystal structures of the isolated intracellular C-linker and CNBD of mammalian HCN1, HCN2, HCN4, and invertebrate spHCN1. These parts of the channel assemble into tetramers (6–9). Further structural information comes from prokaryote ion channels that are homologous to HCN and CNG channels, such as the bacterial cyclic nucleotide-regulated K⁺ channel MloK1 (10–13). MloK1 lacks a C-linker region, but has a CNBD with an overall structure that is remarkably similar to the CNBD of eukaryote HCN channels (10). Based on the dimer assembly of the MloK1 CNBD in the crystal structure, a gating mechanism has been proposed in which the pore opening in the tetrameric channel arises from the action of the four CNBDs as a dimer of dimers (10). The crystal structure of the MloK1 transmembrane domain (11) reveals a domain topology that resembles that of the voltage-gated K⁺ channel Kv1.2 (14), but with important differences. The MloK1 structure suggests that the S1–S4 domain and its associated linker in MloK1 can serve as a clamp to constrain the gate and possibly function in concert with the CNBD to regulate channel opening (11). Additionally, crystal structures have also been determined for the C-linker and cyclic nucleotide binding homology domain (CNBHD) of related ion channels, including the zebrafish EAG-like (ELK) K⁺ channel (15), the mosquito ERG K⁺ channel (16), and the mouse EAG1 K⁺ channel (17). Structural insight into the mechanism of ion permeation has been derived from a prokaryote ion channel NaK (18), which was mutated to mimic the CNG channel pore region (19). Collectively, these structural data have brought valuable information about the determinants of ion permeation, domain assembly, ligand recognition, channel gating and regulation, as well as effects of disease-causing mutations (20).Despite this tremendous progress, crystal structures for whole-eukaryote HCN and CNG channels are still not available at present, and structural insight into fundamental aspects of ion channel function is still lacking, such as the inverse voltage sensitivity in HCN channels and the coupling between cyclic nucleotide binding and channel opening by the C-linker domain, which is, as mentioned, absent in the MloK1 channel (10). In contrast, a putative voltage-gated K⁺ channel containing a C-linker region and CNBD similar to eukaryote channels was identified in the genome of the cyanobacterium Trichodesmium erythraeum (21), here termed TerK, and it was suggested to possibly represent an ancestral HCN or CNG channel (21). However, neither structure nor function of this prokaryote homolog is known. In this study, we report the characterization of TerK and four additional prokaryote ion channels, which all contain six putative transmembrane domains, a C-linker region, and a CNBD, and apparently form a family of prokaryote ion channels with close similarity to eukaryote HCN and CNG channels. We describe the expression in Escherichia coli, detergent screening and biochemical purification of these different homologs. Moreover, we identified two homologs, SthK (from Spirochaeta thermophila) and AmaK (from Arthrospira maxima), which can be stably extracted with detergents and purified in sufficiently high amounts for biochemical and structural studies. Using confocal fluorescence microscopy and electrophysiological recordings, we describe essential functional properties of one homolog, SthK. We find that SthK has electrophysiological properties that closely resemble those of eukaryote CNG channels as it is gated by intracellular cAMP and produces large unitary currents, whereas its activity is relatively insensitive to voltage. However, unlike CNG channels, SthK contains the selectivity filter sequence -TIGYGD-, which is more similar to HCN channels and other K⁺ selective channels. We could experimentally demonstrate that SthK channels are highly selective for K⁺ over Na⁺ ions. Importantly, SthK has several sequence features that closely resemble eukaryote cyclic nucleotide-modulated channels, including a C-linker region, which is missing in previously studied prokaryote homologs, such as MloK1 (10, 12, 13) and MmaK (22). Together, these data make the SthK channel a promising candidate for future structural analysis to learn more about how mammalian CNG and HCN channels work.