Probing the channel-bound shaker B inactivating peptide by stereoisomeric substitution at a strategic tyrosine residue

A synthetic peptide patterned after the sequence of the inactivating ball domain of the Shaker B K(+) channel, the ShB peptide, fully restores fast inactivation in the deletion Shaker BDelta6-46 K(+) channel, which lacks the constitutive ball domains. On the contrary, a similar peptide in which tyrosine 8 is substituted by the secondary structure-disrupting d-tyrosine stereoisomer does not. This suggests that the stereoisomeric substitution prevents the peptide from adopting a structured conformation when bound to the channel during inactivation. Moreover, characteristic in vitro features of the wild-type ShB peptide such as the marked propensity to adopt an intramolecular beta-hairpin structure when challenged by anionic phospholipid vesicles, a model target mimicking features of the inactivation site in the channel protein, or to insert into their hydrophobic bilayers, are lost in the d-tyrosine-containing peptide, whose behavior is practically identical to that of noninactivating peptide mutants. In the absence of high resolution crystallographic data on the inactivated channel/peptide complex, these latter findings suggest that the structured conformation required for the peptide to promote channel inactivation, as referred to above, is likely to be beta-hairpin.     

Structure of the inactivating gate from the Shaker voltage gated K+ channel analyzed by NMR spectroscopy

Rapid inactivation of voltage-gated K⁺ (K_V) channels is mediated by an N-terminal domain (inactivating ball domain) which blocks the open channel from the cytoplasmic side. Inactivating ball domains of various K_V channels are also biologically active when synthesized separately and added as a peptide to the solution. Synthetic inactivating ball domains from different K_V channels with hardly any sequence homology mediate quite similar effects even on unrelated K_V channel subtypes whose inactivation domain has been deleted. The solution structure of the inactivating ball peptide from Shaker (Sh-P22) was analyzed with NMR spectroscopy. The NMR data indicate a non-random structure in an aqueous environment. However, while other inactivating ball peptides showed well-defined three-dimensional structures under these conditions, Sh-P22 does not have a unique, compactly folded structure in solution.     

Interaction between ion channel-inactivating peptides and anionic phospholipid vesicles as model targets.

Studies of rapid (N-type) inactivation induced by different synthetic inactivating peptides in several voltage-dependent cation channels have concluded that the channel inactivation "entrance" (or "receptor" site for the inactivating peptide) consists of a hydrophobic vestibule within the internal mouth of the channel, separated from the cytoplasm by a region with a negative surface potential. These protein domains are conformed from alternative sequences in the different channels and thus are relatively unrestricted in terms of primary structure. We are reporting here on the interaction between the inactivating peptide of the Shaker B K+ channel (ShB peptide) or the noninactivating ShB-L7E mutant with anionic phospholipid vesicles, a model target that, as the channel's inactivation "entrance," contains a hydrophobic domain (the vesicle bilayer) separated from the aqueous media by a negatively charged vesicle surface. When challenged by the anionic phospholipid vesicles, the inactivating ShB peptide 1) binds to the vesicle surface with a relatively high affinity, 2) readily adopts a strongly hydrogen-bonded beta-structure, likely an intramolecular beta "hairpin," and 3) becomes inserted into the hydrophobic bilayer by its folded N-terminal portion, leaving its positively charged C-terminal end exposed to the extravesicular aqueous medium. Similar experiments carried out with the noninactivating, L7E-ShB mutant peptide show that this peptide 1) binds also to the anionic vesicles, although with a lower affinity than does the ShB peptide, 2) adopts only occasionally the characteristic beta-structure, and 3) has completely lost the ability to traverse the anionic interphase at the vesicle surface and to insert into the hydrophobic vesicle bilayer. Because the negatively charged surface and the hydrophobic domains in the model target may partly imitate those conformed at the inactivation "entrance" of the channel proteins, we propose that channel inactivation likely includes molecular events similar to those observed in the interaction of the ShB peptide with the phospholipid vesicles, i.e., binding of the peptide to the region of negative surface potential, folding of the bound peptide as a beta-structure, and its insertion into the channel's hydrophobic vestibule. Likewise, we relate the lack of channel inactivation seen with the mutant ShB-L7E peptide to the lack of ability shown by this peptide to cross through the anionic interphase and insert into the hydrophobic domains of the model vesicle target.     

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" peptide: mapping the inactivating peptide-binding epitope

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.     

Adoption of beta structure by the inactivating "ball" peptide of the Shaker B potassium channel.

The conformation of the inactivating peptide of the Shaker B K+ channel (ShB peptide) and that of a noninactivating mutant (ShBL7E peptide) have been studied. Under all experimental conditions explored, the mutant peptide remains in a predominantly nonordered conformation. On the contrary, the inactivating ShB peptide has a great tendency to adopt a highly stable beta structure, particularly when challenged "in vitro" by anionic phospholipid vesicles. Because the putative peptide binding elements at the inner mouth of the channel comprise a ring of anionic residues and a hydrophobic pocket, we hypothesize that the conformational restrictions imposed on the ShB peptide by its interaction with the anionic lipid vesicles could partly imitate those imposed by the above ion channel elements. Thus, we propose that adoption of beta structure by the inactivating peptide may also occur during channel inactivation. Moreover, the difficulties encountered by the noninactivating ShBL7E peptide mutant to adopt beta structure and the observation that trypsin hydrolysis of the ShB peptide prevent both structure formation and channel inactivation lend further support to the hypothesis that adoption of beta structure by the inactivating peptide in a hydrophobic environment is important in determining channel blockade.     

Solution structure and function of the "tandem inactivation domain" of the neuronal A-type potassium channel Kv1.4

Cumulative inactivation of voltage-gated (Kv) K(+) channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.     

Functional role of the NH2-terminal cytoplasmic domain of a mammalian A- type K channel

It has been shown for a Shaker channel (H-4) that its NH2-terminal cytoplasmic domain may form a "ball and chain" structure, with the "chain" tethering the "ball" to the channel while the "ball" capable of binding to the channel in its open state and causing inactivation. Equivalent structures have not been identified in mammalian Shaker homologues. We studied the functional role of the NH2-terminal region of a fast-inactivating mammalian K channel, RHK1 (Kv1.4), by deleting different domains in this region and examining the resultant changes in channel properties at whole cell and single channel levels. Deleting the NH2-terminal hydrophobic domain (domain A) or the subsequent positive charges (domain I) from RHK1 greatly slowed the decay of whole cell currents, suggesting the existence of a ball-like structure in RHK1 similar to that in the Shaker channel. The function of the ball appeared to be abolished by deleting domain A, while modified but maintained by deleting domain I. In the latter case, the data suggest that the positive charges needed for the function of the ball can be replaced by amino acids from a following region (domain III) that has a high positive charge density. Deleting multiple domains from the NH2 terminus of RHK1 corresponding to the chain in Shaker H-4 did not induce expected changes in channel properties that might result from a shortening of a chain. A comparison of single channel kinetics of selected mutant channels with those of the wild-type channel indicated that these deletion mutations slowed whole cell currents by prolonging burst durations and by increasing the probability of reopening during depolarization. There were no changes in single channel current amplitude or latency to first opening. In conclusion, our observations indicate that the inactivation mechanism of RHK1 is similar to that of Shaker H-4 in that a positively charged cytoplasmic domain is important for such a process. The NH2-terminal domain is not involved in channel activation or ion permeation process.     

Inactivation in ShakerB K+ channels: a test for the number of inactivating particles on each channel.

Fast inactivation in ShakerB K channels results from pore-block caused by "ball peptides" attached to the inner part of each K channel. We have examined the question of how many functional inactivating balls are on each channel and how this number affects inactivation and recovery from inactivation. To that purpose we expressed ShakerB in the insect cell line Sf9 and gradually removed inactivation by perfusing the cell interior with the hydrolytic enzyme papain under whole cell patch clamp. Inactivation slows down as the balls are removed by an amount consistent with the presence of four balls on each channel. Recovery from inactivation has the same time course early and late in papain action; it does not depend on the number of balls remaining on the channel, consistent with the idea that reinactivation is not significant during recovery from inactivation. Our conclusion is that ShakerB has four ball peptides, each capable of causing inactivation. Statistically, the balls are identical and independent. The stability of N-type inactivation by the remaining balls is not appreciably affected by removing some of the balls from a channel.     

Modeling of single noninactivating Na+ channels: evidence for two open and several fast inactivated states

Voltage-gated Na(+) channels play a fundamental role in the excitability of nerve and muscle cells. Defects in fast Na(+) channel inactivation can cause hereditary muscle diseases with hyper- or hypoexcitability of the sarcolemma. To explore the kinetics and gating mechanisms of noninactivating muscle Na(+) channels on a molecular level, we analyzed single channel currents from wild-type and five mutant Na(+) channels. The mutations were localized in different protein regions which have been previously shown to be important for fast inactivation (D3-D4-linker, D3/S4-S5, D4/S4-S5, D4/S6) and exhibited distinct grades of defective fast inactivation with varying levels of persistent Na(+) currents caused by late channel reopenings. Different gating schemes were fitted to the data using hidden Markov models with a correction for time interval omission and compared statistically. For all investigated channels including the wild-type, two open states were necessary to describe our data. Whereas one inactivated state was sufficient to fit the single channel behavior of wild-type channels, modeling the mutants with impaired fast inactivation revealed evidence for several inactivated states. We propose a single gating scheme with two open and three inactivated states to describe the behavior of all five examined mutants. This scheme provides a biological interpretation of the collected data, based on previous investigations in voltage-gated Na(+) and K(+) channels.     

A random flight chain model for the tether of the Shaker K+ channel inactivation domain.

Rapid inactivation of Shaker K+ channels occurs when a domain in the amino terminal region of the channel protein blocks the pore. Some part of the sequence between the inactivating domain and the first transmembrane segment may form a flexible tether. We consider the possibility that the tether has no secondary structure, but is rather a polypeptide random coil. The local concentration of the tethered inactivation domain and the dependence of the inactivation rate on chain length can then be calculated by using the Jacobson-Stockmayer equation. A chain of 30-100 amino acids is consistent with the sensitivity of the inactivation rate to chain length mutations.     

Regulation of N- and C-type inactivation of Kv1.4 by pHo and K+: evidence for transmembrane communication

Kv1.4 encodes a slowly recovering transient outward current (I(to)), which inactivates by a fast N-type (intracellular ball and chain) mechanism but has slow recovery due to C-type inactivation. C-type inactivation of the NH(2)-terminal deletion mutant (fKv1.4DeltaN) was inhibited by 98 mM extracellular K(+) concentration ([K(+)](o)), whereas N-type was unaffected. In 98 mM [K(+)](o), removal of intracellular K(+) concentration ([K(+)](i)) speeded C-type inactivation but had no effect on N-type inactivation, suggesting that C-type inactivation is sensitive to K(+) binding to intracellular sites. C-type inactivation is thought to involve closure of the extracellular pore mouth. However, a valine to alanine mutation on the intracellular side of S6 (V561A) of fKv1.4DeltaN alters recovery and results in anomalous speeding of C-type inactivation with increasing [K(+)](o). Extracellular pH (pH(o)) modulated both N- and C-type inactivation through an S5-H5 linker histidine (H508) with acidosis speeding both N- and C-type inactivation. Mutation of an extracellular lysine to a tyrosine (K532Y) slowed C-type inactivation and inhibited the pH dependence of both N- and C-type inactivation. These results suggest that mutations, [K(+)], and pH modulate inactivation through membrane-spanning mechanisms involving S6.     

Modes of operation of the BKCa channel beta2 subunit

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.     

A new mode of regulation of N-type inactivation in a Caenorhabditis elegans voltage-gated potassium channel

N-type inactivation in voltage-gated K+ (Kv) channels is a widespread means to modulate neuronal excitability and signaling. Here we have shown a novel mechanism of N-type inactivation in a Caenorhabditis elegans Kv channel. The N-terminal sequence of KVS-1 contains a domain of 22 amino acids that resembles the inactivation ball in A-type channels, which is preceded by a domain of eighteen amino acids. Wild type KVS-1 currents can be described as A-type; however, their kinetics are significantly (approximately 5-fold) slower. When the putative inactivation ball is deleted, the current becomes non-inactivating. Inactivation is restored in non-inactivating channels by diffusion of the missing inactivation domain in the cytoplasm. Deletion of the domain in front of the ball speeds inactivation kinetics approximately 5-fold. We conclude that KVS-1 is the first example of a novel type of Kv channel simultaneously possessing an N-inactivating ball preceded by an N inactivation regulatory domain (NIRD) that acts to slow down inactivation through steric mechanisms.     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

Functional role of the N-terminus of Na(+),K(+)-ATPase alpha-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na(+),K(+)-ATPase alpha-subunit shows some homology to that of Shaker-B K(+) channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na(+),K(+)-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the alpha-subunit appears to act like an inactivation gate and performs a critical step in the Na(+),K(+)-ATPase pumping function.     

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.     

Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.

The "shell" model for Ca2(+)-inactivation of Ca2+ channels is based on the accumulation of Ca2+ in a macroscopic shell beneath the plasma membrane. The shell is filled by Ca2+ entering through open channels, with the elevated Ca2+ concentration inactivating both open and closed channels at a rate determined by how fast the shell is filled. In cells with low channel density, the high concentration Ca2+ "shell" degenerates into a collection of nonoverlapping "domains" localized near open channels. These domains form rapidly when channels open and disappear rapidly when channels close. We use this idea to develop a "domain" model for Ca2(+)-inactivation of Ca2+ channels. In this model the kinetics of formation of an inactivated state resulting from Ca2+ binding to open channels determines the inactivation rate, a mechanism identical with that which explains single-channel recordings on rabbit-mesenteric artery Ca2+ channels (Huang Y., J. M. Quayle, J. F. Worley, N. B. Standen, and M. T. Nelson. 1989. Biophys. J. 56:1023-1028). We show that the model correctly predicts five important features of the whole-cell Ca2(+)-inactivation for mouse pancreatic beta-cells (Plants, T. D. 1988. J. Physiol. 404:731-747) and that Ca2(+)-inactivation has only minor effects on the bursting electrical activity of these cells.     

Interaction between fast and ultra-slow inactivation in the voltage-gated sodium channel. Does the inactivation gate stabilize the channel structure?

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na(+) channel mu(1) (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of approximately 100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant mu-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na(+) channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain beta(1) subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the beta(1) subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the beta(1) subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).     

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K⁺. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.