Molecular mechanism for depolarization-induced modulation of Kv channel closure

Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K⁺ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K⁺ conduction through constriction of the K⁺ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history.     

The Link between Ion Permeation and Inactivation Gating of Kv4 Potassium Channels

Kv4 potassium channels undergo rapid inactivation but do not seem to exhibit the classical N-type and C-type mechanisms present in other Kv channels. We have previously hypothesized that Kv4 channels preferentially inactivate from the preopen closed state, which involves regions of the channel that contribute to the internal vestibule of the pore. To further test this hypothesis, we have examined the effects of permeant ions on gating of three Kv4 channels (Kv4.1, Kv4.2, and Kv4.3) expressed in Xenopus oocytes. Rb⁺ is an excellent tool for this purpose because its prolonged residency time in the pore delays K⁺ channel closing. The data showed that, only when Rb⁺ carried the current, both channel closing and the development of macroscopic inactivation are slowed (1.5- to 4-fold, relative to the K⁺ current). Furthermore, macroscopic Rb⁺ currents were larger than K⁺ currents (1.2- to 3-fold) as the result of a more stable open state, which increases the maximum open probability. These results demonstrate that pore occupancy can influence inactivation gating in a manner that depends on how channel closing impacts inactivation from the preopen closed state. By examining possible changes in ionic selectivity and the influence of elevating the external K⁺ concentration, additional experiments did not support the presence of C-type inactivation in Kv4 channels.     

Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K⁺ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K⁺ channel ligands can expand in the subunit interface whose residues are less conserved between K⁺ channels and hence may be targets for design of highly desirable subtype-specific K⁺ channel drugs.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel: THE LID MECHANISM*

Voltage-gated K⁺ (Kv) channels are molecular switches that sense membrane potential and in response open to allow K⁺ ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K⁺ with unchanged fidelity. The implication is that the voltage sensitivity of K⁺ channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.     

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.     

Gating currents from neuronal KV7.4 Channels: General features and correlation with the ionic conductance

The K_V7 (KCNQ) subfamily of voltage-gated K⁺ channels consists of five members (K_V7.1- K_V7.5) giving rise to non-inactivating, and slowly activating/deactivating currents mainly expressed in cardiac (K_V7.1) and neuronal (K_V7.2- K_V7.5) tissue. In the present study, using the cut-open oocyte voltage clamp, we studied the relation of the ionic currents from homomeric neuronal Kv7 channels (K_V7.2-K_V7.5) with the gating currents recorded after K⁺ conductance blockade from the same channels. Increasing the recording temperature from 18{degree sign}C to 28{degree sign}C accelerated activation/deactivation kinetics of the ionic currents in all homomeric K_V7 channels (activation Q10s at 0 mV were 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels, respectively), without large changes in currents voltage-dependence; moreover, at 28{degree sign}C, ionic currents carried by K_V7.4 channels also showed a significant increase in their maximal value. Gating currents were only resolved in K_V7.4 and K_V7.5 channels; the size of the ON gating charges at +40 mV was 1.34 ± 0.34 nC for K_V7.4, and 0.79 ± 0.20 nC for K_V7.5. At 28{degree sign}C, K_V7.4 gating currents had the following salient properties: 1) similar time integral of QON and QOFF, indicating no charge immobilization; 2) a left-shift in the V1/2 of the QON/V when compared to the G/V (≈ 50 mV in the presence of 2 mM extracellular Ba²⁺); 3) a QON decay faster than ionic current activation; and 4) a rising phase in the OFF gating charge after depolarizations larger than 0 mV. These observations suggest that, in K_V7.4 channels, VSD movement is followed by a slow and/or low bearing charge step linking to pore opening, a result which may help to clarify the molecular consequence of disease-causing mutations and drugs affecting channel gating.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K+-free solutions at physiological pH

Extracellular acidification and reduction of extracellular K⁺ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K⁺]_o at pH 7.4, it is very sensitive to [K⁺]_o at pH 6.4, and in the mutant, H463G, the removal of K⁺ _o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K⁺ _o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na⁺ permeation through inactivated channels. Although the H463G currents were abolished in zero [K⁺]_o, robust Na⁺ tail currents through inactivated channels were observed. The appearnnce of H463G Na⁺ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K⁺ was replaced by NMG⁺ and the inward Na⁺ current was recorded, addition of 1 mM K⁺ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K⁺]_o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).     

Intracellular domains interactions and gated motions of IKS potassium channel subunits

Voltage‐gated K⁺ channels co‐assemble with auxiliary β subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore‐forming subunits with KCNE1 β subunits generates the repolarizing K⁺ current I_KS. However, the detailed nature of their interface remains unknown. Mutations in either Kv7.1 or KCNE1 produce the life‐threatening long or short QT syndromes. Here, we studied the interactions and voltage‐dependent motions of I_KS channel intracellular domains, using fluorescence resonance energy transfer combined with voltage‐clamp recording and in vitro binding of purified proteins. The results indicate that the KCNE1 distal C‐terminus interacts with the coiled‐coil helix C of the Kv7.1 tetramerization domain. This association is important for I_KS channel assembly rules as underscored by Kv7.1 current inhibition produced by a dominant‐negative C‐terminal domain. On channel opening, the C‐termini of Kv7.1 and KCNE1 come close together. Co‐expression of Kv7.1 with the KCNE1 long QT mutant D76N abolished the K⁺ currents and gated motions. Thus, during channel gating KCNE1 is not static. Instead, the C‐termini of both subunits experience molecular motions, which are disrupted by the D76N causing disease mutation.     

Properties of deactivation gating currents in Shaker channels

The charge versus voltage relation of voltage-sensor domains shifts in the voltage axis depending on the initial voltage. Here we show that in nonconducting W434F Shaker K⁺ channels, a large portion of this charge-voltage shift is apparent due to a dramatic slowing of the deactivation gating currents, Ig_D (with τ up to 80 ms), which develops with a time course of ∼1.8 s. This slowing in Ig_D adds up to the slowing due to pore opening and is absent in the presence of 4-aminopyridine, a compound that prevents the last gating step that leads to pore opening. A remaining 10–15 mV negative shift in the voltage dependence of both the kinetics and the charge movement persists independently of the depolarizing prepulse duration and remains in the presence of 4-aminopyridine, suggesting the existence of an intrinsic offset in the local electric field seen by activated channels. We propose a new (to our knowledge) kinetic model that accounts for these observations.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

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.     

Mixed modes in opening of KcsA potassium channel from a targeted molecular dynamics simulation

Potassium channels conduct K⁺ flow selectively across the membrane through a central pore. During a process called gating, the potassium channels undergo a conformational change that opens or closes the ion-conducting pore. The potassium channel KcsA has been structurally determined in its closed state. However, the dynamic mechanism of the gating transition of the KcsA channel is still being investigated. Here, a targeted molecular dynamics simulation up to 150 ns is performed to investigate the detailed opening process of the KcsA channel with an open Kv1.2 structure serving as the target. The channel arrived at a self-determined quasi-stable state within 60 ns. The rigid-body and hinge-bending modes are observed mixed together in the remaining 90 ns long quasi-stable state. The mixed-mode movement seems come from the competition between the helix rigidity and the biased-applied gating force.     

Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels

K channels mediate the selective passage of K⁺ across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K⁺ transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker’s reported ∼20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K⁺ is set to ∼4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K⁺], beyond that of P475D, suggesting an ∼200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K⁺ transport rates to ∼1 kT. Thus, although Shaker’s pore sustains ion translocation as the BK channel’s does, higher energetic costs of ion stabilization or higher friction with the ion’s rigid hydration cage in its narrower aqueous cavity may entail higher resistance.     

Differential expression of the Kv1 voltage-gated potassium channel family in the rat nephron

Several potassium (K⁺) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K⁺ channels allow sodium reabsorption in the proximal tubule (PT), K⁺ recycling and K⁺ reabsorption in the thick ascending limb (TAL) and K⁺ secretion and K⁺ reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K⁺ to function as a secretory K⁺ channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K⁺ channels. Our results expand the repertoire of K⁺ channels that contribute to K⁺ homeostasis to include the Kv1 family.     

Divalent Cations Slow Activation of EAG Family K Channels through Direct Binding to S4

Voltage-gated K⁺ channels share a common voltage sensor domain (VSD) consisting of four transmembrane helices, including a highly mobile S4 helix that contains the major gating charges. Activation of ether-a-go-go (EAG) family K⁺ channels is sensitive to external divalent cations. We show here that divalent cations slow the activation rate of two EAG family channels (Kv12.1 and Kv10.2) by forming a bridge between a residue in the S4 helix and acidic residues in S2. Histidine 328 in the S4 of Kv12.1 favors binding of Zn²⁺ and Cd²⁺, whereas the homologous residue Serine 321 in Kv10.2 contributes to effects of Mg²⁺ and Ni²⁺. This novel finding provides structural constraints for the position of transmembrane VSD helices in closed, ion-bound EAG family channels. Homology models of Kv12.1 and Kv10.2 VSD structures based on a closed-state model of the Shaker family K⁺ channel Kv1.2 match these constraints. Our results suggest close conformational conservation between closed EAG and Shaker family channels, despite large differences in voltage sensitivity, activation rates, and activation thresholds.