Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

Structure and function of potassium channels in plants: some inferences about the molecular origin of inward rectification in KAT1 channels (Review)

Potassium channels in plants play a variety of important physiological roles including K⁺ uptake into roots, stomatal and leaf movements, and release of K⁺ into the xylem. This review summarizes current knowledge about a class of plant genes whose products are K⁺ channel-forming proteins. Potassium channels of this class belong to a superfamily characterized by six membrane-spanning domains (S1-6), a positively charged S4 domain and a region between the S5 and S6 segments that forms the channel selectivity filter. These channels are voltage dependent, which means the membrane potential modifies the probability of opening (P_o). However, despite these channels sharing the same topology as the outward-rectifying K⁺ channels, which are activated by membrane depolarization, some plant K⁺ channels such as KAT1/2 and KST1 open with hyperpolarizing voltages. In outward-rectifying K⁺ channels, the change in P_o is achieved through a voltage sensor formed by the S4 segment that detects the voltage transferring its energy to the gate that controls pore opening. This coupling is achieved by an outward displacement of the charges contained in S4. In KAT1, most of the results indicate that S4 is the voltage sensor. However, how the movement of S4 leads to opening remains unanswered. On the basis of recent data, we propose here that in plant-inward rectifiers an inward movement of S4 leads to channel opening and that the difference between it and outward-rectifying channels resides in the mechanism that couples gating charge displacement with pore opening.     

Flavonoid Regulation of HCN2 Channels

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC₅₀) of 1.8 μm. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.     

A Leucine Zipper Motif Essential for Gating of Hyperpolarization-activated Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are pacemakers in cardiac myocytes and neurons. Although their membrane topology closely resembles that of voltage-gated K⁺ channels, the mechanism of their unique gating behavior in response to hyperpolarization is still poorly understood. We have identified a highly conserved leucine zipper motif in the S5 segment of HCN family members. In order to study the role of this motif for channel function, the leucine residues of the zipper were individually mutated to alanine, arginine, or glutamine residues. Leucine zipper mutants traffic to the plasma membrane, but the channels lose their sensitivity to open upon hyperpolarization. Thus, our data indicate that the leucine zipper is an important molecular determinant for hyperpolarization-activated channel gating. Residues of the leucine zipper interact with the adjacent S6 segment of the channel. This interaction is essential for voltage-dependent gating of the channel. The lower part of the leucine zipper, at the intracellular mouth of the channel, is important for stabilizing the closed state. Mutations at these sites increase current amplitudes or result in channels with deficient closing and increased min-P_o. Our data are further supported by homology models of the open and closed state of the HCN2 channel pore. Thus, we conclude that the leucine zipper of HCN channels is a major determinant for hyperpolarization-activated channel gating.     

Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the K_v1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.     

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate (PIP₂) regulates Shaker K⁺ channels and voltage-gated Ca²⁺ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP₂ regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP₂ regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K⁺ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP₂ regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K⁺ channel family. Open-state stabilization by PIP₂ has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP₂ strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP₂ has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4–S5 linker) and R479 near the S6 activation gate are required for PIP₂ to inhibit voltage activation. The ability of PIP₂ to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP₂ on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP₂-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP₂ can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP₂ regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.     

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K⁺) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb⁺ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG⁺), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K⁺ (2.66 Å in diameter). However, in the absence of K⁺, significant NMDG⁺ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg²⁺ (1 mM), which blocked the outward NMDG⁺ current, resulting in a strong inward rectification. The NMDG⁺ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG⁺ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K⁺ added to the external NMDG⁺ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K⁺ over NMDG⁺ (P_K⁺/P_NMDG⁺) of ∼240. Reversal potential shifts in mixtures of K⁺ and NMDG⁺ are in accordance with P_K⁺/P_NMDG⁺, indicating that the ions compete for permeation and suggesting that NMDG⁺ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG⁺, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.     

Properties of inwardly rectifying K+ channels in ventricular myocytes

Summary The voltage- and time-dependent properties of whole-cell, multi-channel (outside-out), and single channel inwardly-rectifying K⁺ currents were studied using adult and neonatal rat, and embryonic chick ventricular myocytes. Inward rectification of the current-voltage relationship was found in the whole-cell and single channel measurements. The steady-state single channel probability of opening decreased with hyperpolarization from E_K, as did the mean open time, thereby explaining the time-dependent inactivation of the macroscopic current. Myocytes dialysed with a Mg⁺⁺-free K⁺ solution (to remove the property of inward rectification) displayed a quasi-linear current-voltage relationship. The outward K⁺ currents flowing through the modified inward rectifier channels were able to be blocked by the local anesthetic and anti-arrhythmic agent, lidocaine.     

Intersubunit Coupling in the Pore of BK Channels

The structural basis underlying the gating of large conductance Ca²⁺-activated K⁺ (BK) channels remains elusive. We found that substitution of Leu-312 in the S6 transmembrane segment of mSlo1 BK channels with hydrophilic amino acids of smaller side-chain volume favored the open state. The sensitivities of channels to calcium and voltage were modified by some mutations and completely abolished by others. Interpretation of the results in terms of an allosteric model suggests that the calcium-insensitive mutants greatly destabilize the closed relative to the open conformation and may also disrupt the allosteric coupling between Ca²⁺ or voltage sensors and the gate. Some Phe-315 mutations also favor the open state, suggesting that Leu-312 and Phe-315 may interact in the closed state, forming a major energy barrier that the channel has to overcome to open. Homology modeling and molecular dynamic simulations further support that the side chain of Leu-312 can couple strongly with the aromatic ring of Phe-315 in neighboring subunits (L-F coupling) to maintain the channel closed. Additionally, single-channel recordings indicate that the calcium-insensitive mutants, whose kinetics can be approximately characterized by a two-state closed-open (C-O) model, exhibit nearly 100% open probability under physiological conditions without alterations in single-channel conductance. These findings provide a basis for understanding the structure and gating of the BK channel pore.High conductance, “big” K⁺ (BK)⁴ channels encoded by the Slo1 gene are widely expressed in many tissues and respond to both membrane depolarization and submembrane Ca²⁺ concentration ([Ca²⁺]_i) (1–4). Because BK channels have apparently evolved from voltage-dependent K⁺ (K_V) channels, they share many features in common with K_V channels such as a similar K⁺ selectivity filter and conserved ion conduction pore (5). On the other hand, detailed studies indicate that there are significant differences in the permeation and gating properties of K_V and BK channels. First, BK channels have the largest single-channel conductance among K⁺ channels under similar recording conditions (6), partly because of two negatively charged rings at the entrance to the intracellular vestibule of BK channels that are absent from many K_V channels (7). Second, the mouth of the BK pore on the cytoplasmic side appears to be larger than other K⁺ channels because the on-rates for channel block by quaternary ammonium (QA) ions are limited only by diffusion (8), whereas the on-rates for QA block of other K⁺ channels are substantially slower. Additionally, it has been proposed that the pore-lining S6 segment of BK channels may not act as a permeation gate (8) as in Shaker K_V channels (9). Therefore, the BK channel may have an unusual pore, and understanding the structural basis of the above differences could provide important insight into the permeation and gating properties of K⁺ channels.Despite the differences between BK and K_V channels, it is likely that the opening and closing of their pores involve similar conformational changes. Flexing of the inner S6 helix at or near a conserved glycine residue is thought to open the pore, whereas straightening closes it (10, 11). The inner helix of BK channels is potentially more flexible than that of K_V channels because it contains a twin glycine motif (Gly-310–Gly-311). Differences in permeation property, pore geometry, and gating could potentially be accounted for by differences in S6 flexibility as well as the specific side-chain properties and interactions that occur between pore-lining residues. In this study we focused on Leu-312 and Phe-315 because we inferred from sequence analogy with KcsA channels (12) and our previous studies of BK channels (13, 14) that they are two of the five pore-lining residues (the others being Leu-309, Val-319, and Ile-323), and they are near the putative gating hinge. Our results indicate that substitutions of Leu-312 or Phe-315 have profound effects on BK channel gating, consistent with the notion that these residues interact between different subunits to hold the channel closed. The interaction of these residues may also be important for coordinating conformational changes in different subunits to ensure the efficient and cooperative long-range coupling between channel subunits, which is probably a general property of allosterically regulated proteins. Moreover, a surprising loss of Ca²⁺- and voltage sensitivity that accompanies mutations of Leu-312 or Phe-315 raises the possibility that these residues play a broader role in allosteric communication between sensors and gates.     

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.     

Basis for allosteric open-state stabilization of voltage-gated potassium channels by intracellular cations

The open state of voltage-gated potassium (Kv) channels is associated with an increased stability relative to the pre-open closed states and is reflected by a slowing of OFF gating currents after channel opening. The basis for this stabilization is usually assigned to intrinsic structural features of the open pore. We have studied the gating currents of Kv1.2 channels and found that the stabilization of the open state is instead conferred largely by the presence of cations occupying the inner cavity of the channel. Large impermeant intracellular cations such as N-methyl-d-glucamine (NMG⁺) and tetraethylammonium cause severe slowing of channel closure and gating currents, whereas the smaller cation, Cs⁺, displays a more moderate effect on voltage sensor return. A nonconducting mutant also displays significant open state stabilization in the presence of intracellular K⁺, suggesting that K⁺ ions in the intracellular cavity also slow pore closure. A mutation in the S6 segment used previously to enlarge the inner cavity (Kv1.2-I402C) relieves the slowing of OFF gating currents in the presence of the large NMG⁺ ion, suggesting that the interaction site for stabilizing ions resides within the inner cavity and creates an energetic barrier to pore closure. The physiological significance of ionic occupation of the inner cavity is underscored by the threefold slowing of ionic current deactivation in the wild-type channel compared with Kv1.2-I402C. The data suggest that internal ions, including physiological concentrations of K⁺, allosterically regulate the deactivation kinetics of the Kv1.2 channel by impairing pore closure and limiting the return of voltage sensors. This may represent a primary mechanism by which Kv channel deactivation kinetics is linked to ion permeation and reveals a novel role for channel inner cavity residues to indirectly regulate voltage sensor dynamics.     

KCNQ1 Channels Do Not Undergo Concerted but Sequential Gating Transitions in Both the Absence and the Presence of KCNE1 Protein

The co-assembly of KCNQ1 with KCNE1 produces I_KS, a K⁺ current, crucial for the repolarization of the cardiac action potential. Mutations in these channel subunits lead to life-threatening cardiac arrhythmias. However, very little is known about the gating mechanisms underlying KCNQ1 channel activation. Shaker channels have provided a powerful tool to establish the basic gating mechanisms of voltage-dependent K⁺ channels, implying prior independent movement of all four voltage sensor domains (VSDs) followed by channel opening via a last concerted cooperative transition. To determine the nature of KCNQ1 channel gating, we performed a thermodynamic mutant cycle analysis by constructing a concatenated tetrameric KCNQ1 channel and by introducing separately a gain and a loss of function mutation, R231W and R243W, respectively, into the S4 helix of the VSD of one, two, three, and four subunits. The R231W mutation destabilizes channel closure and produces constitutively open channels, whereas the R243W mutation disrupts channel opening solely in the presence of KCNE1 by right-shifting the voltage dependence of activation. The linearity of the relationship between the shift in the voltage dependence of activation and the number of mutated subunits points to an independence of VSD movements, with each subunit incrementally contributing to channel gating. Contrary to Shaker channels, our work indicates that KCNQ1 channels do not experience a late cooperative concerted opening transition. Our data suggest that KCNQ1 channels in both the absence and the presence of KCNE1 undergo sequential gating transitions leading to channel opening even before all VSDs have moved.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

Transferring knowledge towards understanding the pore stabilizing variations in K+ channels

Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K⁺ channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K⁺ channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K⁺ channels.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

A Mechanism for the Auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Opening and Its Relief by cAMP

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1–4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic “C-linker,” which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V_½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or “tetrameric” C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated.     

Complex interactions among residues within pore region determine the K+ dependence of a KAT1‐type potassium channel AmKAT1

KAT1‐type channels mediate K⁺ influx into guard cells that enables stomatal opening. In this study, a KAT1‐type channel AmKAT1 was cloned from the xerophyte Ammopiptanthus mongolicus. In contrast to most KAT1‐type channels, its activation is strongly dependent on external K⁺ concentration, so it can be used as a model to explore the mechanism for the K⁺‐dependent gating of KAT1‐type channels. Domain swapping between AmKAT1 and KAT1 reveals that the S5–pore–S6 region controls the K⁺ dependence of AmKAT1, and residue substitutions show that multiple residues within the S5–Pore linker and Pore are involved in its K⁺‐dependent gating. Importantly, complex interactions occur among these residues, and it is these interactions that determine its K⁺ dependence. Finally, we analyzed the potential mechanism for the K⁺ dependence of AmKAT1, which could originate from the requirement of K⁺ occupancy in the selectivity filter to maintain its conductive conformation. These results provide new insights into the molecular basis of the K⁺‐dependent gating of KAT1‐type channels.     

Characterization of the Driving Force as a Modulator of Gating in Cardiac ATP-sensitive K+ Channels — Evidence for Specific Elementary Properties

Single cardiac ATP-sensitive K⁺ channels and, comparatively, two other members of the inwardly rectifying K⁺ channel family, cardiac K⁺ _(ir) and K⁺ _(ACh) channels, were studied in the inside-out recording mode in order to analyze influence and significance of the electrochemical K⁺ gradient for open-state kinetics of these K⁺ channels. The conductive state of K⁺ _(ATP) channels was defined as a function of the electrochemical K⁺ gradient in that increased driving force correlates with shortened open-channel lifetime. Flux coupling of gating can be largely excluded as the underlying mechanism for two reasons: (i) τ_open proved identical in 23 pS, 56 pS and 80 pS channels; (ii) K⁺ _(ATP) channel protonation by an external pH shift from 9.5 to 5.5 reduced conductance without a concomitant detectable change of τ_open. Since gating continued to operate at E _K , i.e., in the absence of K⁺ permeation through the pore, K⁺ driving force cannot be causally involved in gating. Rather the driving force acts to modulate the gating process similar to Rb⁺ whose interference with an externally located binding site stabilizes the open state. In K⁺ _(ir) and K⁺ _(ACh) channels, the open state is essentially independent on driving force meaning that their gating apparatus does not sense the electrochemical K⁺ gradient. Thus, K⁺ _(ATP) channels differ in an important functional aspect which may be tentatively explained by a structural peculiarity of their gating apparatus. Received: 24 March 1997/Revised: 24 April 1998     

Involvement of the S4-S5 linker and the C-linker domain regions to voltage-gating in plant Shaker channels: Comparison with animal HCN and Kv channels

Among the different transport systems present in plant cells, Shaker channels constitute the major pathway for K⁺ in the plasma membrane. Plant Shaker channels are members of the 6 transmembrane-1 pore (6TM-1P) cation channel superfamily as the animal Shaker (Kv) and HCN channels. All these channels are voltage-gated K⁺ channels: Kv channels are outward-rectifiers, opened at depolarized voltages and HCN channels are inward-rectifiers, opened by membrane hyperpolarization. Among plant Shaker channels, we can find outward-rectifiers, inward-rectifiers and also weak-rectifiers, with weak voltage dependence. Despite the absence of crystal structures of plant Shaker channels, functional analyses coupled to homology modeling, mostly based on Kv and HCN crystals, have permitted the identification of several regions contributing to plant Shaker channel gating. In the present mini-review, we make an update on the voltage-gating mechanism of plant Shaker channels which seem to be comparable to that proposed for HCN channels.