Role of Outer-pore Residue Y380 in U-type Inactivation of KV2.1 Channels

The interpretation of slow inactivation in potassium channels has been strongly influenced by work on C-type inactivation in Shaker channels. Slow inactivation in Shaker and some other potassium channels can be dramatically modulated by the state of the pore, including mutations at outer pore residue T449, which altered inactivation kinetics up to 100-fold. K_V2.1, another voltage-dependent potassium channel, exhibits a biophysically distinct inactivation mechanism with a U-shaped voltage-dependence and preferential closed-state inactivation, termed U-type inactivation. However, it remains to be demonstrated whether U-type and C-type inactivation have different molecular mechanisms. This study examines mutations at Y380 (homologous to Shaker T449) to investigate whether C-type and U-type inactivation have distinct molecular mechanisms, and whether C-type inactivation can occur at all in K_V2.1. Y380 mutants do not introduce C-type inactivation into K_V2.1 and have little effect on U-type inactivation of K_V2.1. Interestingly, two of the mutants tested exhibit twofold faster recovery from inactivation compared to wild-type channels. The observation that mutations have little effect suggests K_V2.1 lacks C-type inactivation as it exists in Shaker and that C-type and U-type inactivation have different molecular mechanisms. Kinetic modeling predicts that all mutants inactivate preferentially, but not exclusively, from partially activated closed states. Therefore, K_V2.1 exhibits a single U-type inactivation process including some inactivation from open as well as closed states.     

U-type inactivation of Kv3.1 and Shaker potassium channels.

We previously concluded that the Kv2.1 K(+) channel inactivates preferentially from partially activated closed states. We report here that the Kv3.1 channel also exhibits two key features of this inactivation mechanism: a U-shaped voltage dependence measured at 10 s and stronger inactivation with repetitive pulses than with a single long depolarization. More surprisingly, slow inactivation of the Kv1 Shaker K(+) channel (Shaker B Delta 6--46) also has a U-shaped voltage dependence for 10-s depolarizations. The time and voltage dependence of recovery from inactivation reveals two distinct components for Shaker. Strong depolarizations favor inactivation that is reduced by K(o)(+) or by partial block by TEA(o), as previously reported for slow inactivation of Shaker. However, depolarizations near 0 mV favor inactivation that recovers rapidly, with strong voltage dependence (as for Kv2.1 and 3.1). The fraction of channels that recover rapidly is increased in TEA(o) or high K(o)(+). We introduce the term U-type inactivation for the mechanism that is dominant in Kv2.1 and Kv3.1. U-type inactivation also makes a major but previously unrecognized contribution to slow inactivation of Shaker.     

Molecular determinants of U-type inactivation in Kv2.1 channels

Kv2.1 channels exhibit a U-shaped voltage-dependence of inactivation that is thought to represent preferential inactivation from preopen closed states. However, the molecular mechanisms underlying so-called U-type inactivation are unknown. We have performed a cysteine scan of the S3-S4 and S5-P-loop linkers and found sites that are important for U-type inactivation. In the S5-P-loop linker, U-type inactivation was preserved in all mutant channels except E352C. This mutation, but not E352Q, abolished closed-state inactivation while preserving open-state inactivation, resulting in a loss of the U-shaped voltage profile. The reducing agent DTT, as well as the C232V mutation in S2, restored U-type inactivation to the E352C mutant, which suggests that residues 352C and C232 may interact to prevent U-type inactivation. The R289C mutation, in the S3-S4 linker, also reduced U-type inactivation. In this case, DTT had little effect but application of MTSET restored wild-type-like U-type inactivation behavior, suggestive of the importance of charge at this site. Kinetic modeling suggests that the E352C and R289C inactivation phenotypes largely resulted from reductions in the rate constants for transitions from closed to inactivated states. The data indicate that specific residues within the S3-S4 and S5-P-loop linkers may play important roles in Kv2.1 U-type inactivation.     

Kv Channel Gating Requires a Compatible S4-S5 Linker and Bottom Part of S6, Constrained by Non-interacting Residues

Voltage-dependent K⁺ channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6_T) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6_T. Exchanging both L45 and S6_T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6_T in hKv1.5 further shows that S6_T needs to be α-helical and forms a “crevice” in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6_T crevice, whereas other residues in S6_T and L45 that are not involved in the interaction maintain the correct structure of the coupling.     

Shaker IR T449 Mutants Separate C- from U-Type Inactivation

Previous studies demonstrated that slow inactivation of the Shaker potassium channel can be made ~100-fold faster or slower by point mutations at a site in the outer pore (T449). However, the discovery that two forms of slow inactivation coexist in Shaker raises the question of which inactivation process is affected by mutation. Equivalent mutations in K_V2.1, a channel exhibiting only U-type inactivation, have minimal effects on inactivation, suggesting that mutation of Shaker T449 acts on C-type inactivation alone, a widely held yet untested hypothesis. This study reexamines mutations at Shaker T449, confirming that T449A speeds inactivation and T449Y/V slow it. T449Y and T449V exhibit U-type inactivation that is enhanced by high extracellular potassium, in contrast to C-type inactivation in T449A which is inhibited by high potassium. Automated parameter estimation for a 12-state Markov model suggests that U-type inactivation occurs mainly from closed states upon weak depolarization, but primarily from the open state at positive voltages. The model also suggests that WT channels, which in this study exhibit mostly C-type inactivation, recover from inactivation through closed-inactivated states, producing voltage-dependent recovery. This suggests that both C-type and U-type inactivation involve both open-inactivated and closed-inactivated states.     

N type rapid inactivation in human Kv1.4 channels: functional role of a putative C-terminal helix

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a ‘ball and chain’ mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini.     

N type rapid inactivation in human Kv1.4 channels: functional role of a putative C-terminal helix

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a 'ball and chain' mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini.     

Episodic ataxia type-1 mutations in the hKv1.1 cytoplasmic pore region alter the gating properties of the channel.

Episodic ataxia type-1 is a rare human neurological syndrome which occurs during childhood and persists through the whole life of affected patients. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene hKv1.1 of different affected families. V408A and E325D mutations are located in the cytoplasmic putative pore region of hKv1.1 channels and profoundly alter their gating properties. V408A channels showed increased kinetic rates of activation, deactivation and C-type inactivation. Expression of E325D channels in Xenopus oocytes led to an approximately 13-fold current amplitude reduction and to a 52.4 mV positive shift in the voltage dependence of activation. Moreover, the E325D mutation altered the kinetics of activation, deactivation, C-type inactivation and channel open probability. Heteromeric channels composed of two wild-type and two mutated subunits, linked as dimers, showed gating properties intermediate between channels formed from four normal or four mutated subunits. The results demonstrate that the highly conserved residues Val408 and Glu325 play a pivotal role in several gating processes of a human potassium channel, and suggest a pathogenetic mechanism by which the impairment of the delayed-rectifier function of affected neurons is related to the type and number of mutated subunits which make up the hKv1.1 channels.     

Interaction of the S6 Proline Hinge with N-Type and C-Type Inactivation in?Kv1.4 Channels

Several voltage-gated channels share a proline-valine-proline (proline hinge) sequence motif at the intracellular side of S6. We studied the proline hinge in Kv1.4 channels, which inactivate via two mechanisms: N- and C-type. We mutated the second proline to glycine or alanine: P558A, P558G. These mutations were studied in the presence/absence of the N-terminal to separate the effects of the interaction between the proline hinge and N- and C-type inactivation. Both S6 mutations slowed or removed N- and C-type inactivation, and altered recovery from inactivation. P558G slowed activation and N- and C-type inactivation by nearly an order of magnitude. Sensitivity to extracellular acidosis and intracellular quinidine binding remained, suggesting that transmembrane communication in N- and C-type inactivation was preserved, consistent with our previous findings of major structural rearrangements involving S6 during C-type inactivation. P558A was very disruptive: activation was slowed by more than an order of magnitude, and no inactivation was observed. These results are consistent with our hypothesis that the proline hinge and intracellular S6 movement play a significant role in inactivation and recovery. Computer modeling suggests that both P558G and P558A mutations modify early voltage-dependent steps and make a final voltage-insensitive step that is rate limiting at positive potentials.     

A model of the interaction between N-type and C-type inactivation in Kv1.4 channels

Kv1.4 channels are Shaker-related voltage-gated potassium channels with two distinct inactivation mechanisms. Fast N-type inactivation operates by a ball-and-chain mechanism. Slower C-type inactivation is not so well defined, but involves intracellular and extracellular conformational changes of the channel. We studied the interaction between inactivation mechanisms using two-electrode voltage-clamp of Kv1.4 and Kv1.4ΔN (amino acids 2–146 deleted to remove N-type inactivation) heterologously expressed in Xenopus oocytes. We manipulated C-type inactivation by introducing a lysine-tyrosine point mutation (K532Y, equivalent to Shaker T449Y) that diminishes C-type inactivation. We used experimental data to develop a comprehensive computer model of Kv1.4 channels to determine the interaction between activation and N- and C-type inactivation mechanisms needed to replicate the experimental data. C-type inactivation began at lower voltage preactivated states, whereas N-type inactivation was coupled directly to the open state. A model with distinct N- and C-type inactivated states was not able to reproduce experimental data, and direct transitions between N- and C-type inactivated states were required, i.e., there is coupling between N- and C-type inactivated states. C-type inactivation is the rate-limiting step determining recovery from inactivation, so understanding C-type inactivation, and how it is coupled to N-type inactivation, is critical in understanding how channels act to repetitive stimulation.     

The antibody targeting the E314 peptide of human Kv1.3 pore region serves as a novel, potent and specific channel blocker

Selective blockade of Kv1.3 channels in effector memory T (T(EM)) cells was validated to ameliorate autoimmune or autoimmune-associated diseases. We generated the antibody directed against one peptide of human Kv1.3 (hKv1.3) extracellular loop as a novel and possible Kv1.3 blocker. One peptide of hKv1.3 extracellular loop E3 containing 14 amino acids (E314) was chosen as an antigenic determinant to generate the E314 antibody. The E314 antibody specifically recognized 63.8KD protein stably expressed in hKv1.3-HEK 293 cell lines, whereas it did not recognize or cross-react to human Kv1.1(hKv1.1), Kv1.2(hKv1.2), Kv1.4(hKv1.4), Kv1.5(hKv1.5), KCa3.1(hKCa3.1), HERG, hKCNQ1/hKCNE1, Nav1.5 and Cav1.2 proteins stably expressed in HEK 293 cell lines or in human atrial or ventricular myocytes by Western blotting analysis and immunostaining detection. By the technique of whole-cell patch clamp, the E314 antibody was shown to have a directly inhibitory effect on hKv1.3 currents expressed in HEK 293 or Jurkat T cells and the inhibition showed a concentration-dependence. However, it exerted no significant difference on hKv1.1, hKv1.2, hKv1.4, hKv1.5, hKCa3.1, HERG, hKCNQ1/hKCNE1, L-type Ca(2+) or voltage-gated Na(+) currents. The present study demonstrates that the antibody targeting the E314 peptide of hKv1.3 pore region could be a novel, potent and specific hKv1.3 blocker without affecting a variety of closely related K(v)1 channels, KCa3.1 channels and functional cardiac ion channels underlying central nervous system (CNS) disorders or drug-acquired arrhythmias, which is required as a safe clinic-promising channel blocker.     

The electrically silent Kv6.4 subunit confers hyperpolarized gating charge movement in Kv2.1/Kv6.4 heterotetrameric channels

The voltage-gated K(+) (Kv) channel subunit Kv6.4 does not form functional homotetrameric channels but co-assembles with Kv2.1 to form functional Kv2.1/Kv6.4 heterotetrameric channels. Compared to Kv2.1 homotetramers, Kv6.4 exerts a ~40 mV hyperpolarizing shift in the voltage-dependence of Kv2.1/Kv6.4 channel inactivation, without a significant effect on activation gating. However, the underlying mechanism of this Kv6.4-induced modulation of Kv2.1 channel inactivation, and whether the Kv6.4 subunit participates in the voltage-dependent gating of heterotetrameric channels is not well understood. Here we report distinct gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels, compared to Kv2.1 homotetramers, as revealed by gating current recordings from mammalian cells expressing these channels. The gating charge movement of Kv2.1/Kv6.4 heterotetrameric channels displayed an extra component around the physiological K(+) equilibrium potential, characterized by a second sigmoidal relationship of the voltage-dependence of gating charge movement. This distinct gating charge displacement reflects movement of the Kv6.4 voltage-sensing domain and has a voltage-dependency that matches the hyperpolarizing shift in Kv2.1/Kv6.4 channel inactivation. These results provide a mechanistic basis for the modulation of Kv2.1 channel inactivation gating kinetics by silent Kv6.4 subunits.     

Mutations in the KCNA1 gene associated with episodic ataxia type-1 syndrome impair heteromeric voltage-gated K(+) channel function.

Episodic ataxia type-1 syndrome (EA-1) is an autosomal dominant neurological disorder that manifests itself during infancy and results from point mutations in the voltage-gated potassium channel gene hKv1.1. The hallmark of the disease is continuous myokymia and episodic attacks of spastic contractions of the skeletal muscles, which cause permanent disability. Coexpression of hKv1.1 and hKv1.2 subunits produces heteromeric potassium channels with biophysical and pharmacological properties intermediate between the respective homomers. By using tandemly linked subunits, we demonstrate that hKv1.1 subunits bearing the EA-1 mutations V408A and E325D combine with hKv1.2 to produce channels with altered kinetics of activation, deactivation, C-type inactivation, and voltage dependence. Moreover, hKv1.1V408A single-channel analysis reveals a approximately threefold reduction of the mean open duration of the channel compared with the wild-type, and this mutation alters the open-state stability of both homomeric and heteromeric channels. The results demonstrate that human Kv1.2 and Kv1.1 subunits coassemble to form a novel channel with distinct gating properties that are altered profoundly by EA-1 mutations, thus uncovering novel physiopathogenetic mechanisms of episodic ataxia type-1 myokymia syndrome.     

External nickel blocks human Kv1.5 channels stably expressed in CHO cells

We have investigated the actions of Nickel (Ni²⁺) on a human cardiac potassium channel (hKv1.5), the main component of human atrial ultra-rapid delayed rectifier current, stably expressed in Chinese hamster ovary cell line using the whole-cell voltage-clamp technique. External Ni²⁺ reversibly decreased the amplitude of the current in a concentration-dependent manner. The concentration for half-maximum inhibition of the current at +50 mV was 568 μm. The activation, deactivation, reactivation kinetics of the current were not affected by Ni²⁺. Block was not voltage-dependent but frequency-dependent block was apparent. The extent of channel block during the first pulse increased when the duration of exposure to Ni²⁺, prior to channel activation, was prolonged indicating that Ni²⁺ interacted with hKv1.5 in the closed state. The percentage of current remaining in presence of Ni²⁺ decreased steeply over the range of steady-state channel inactivation, consistent with an enhanced block with increased inactivation. This suggests that Ni²⁺ preferentially blocks nonconducting hKv1.5 channels, either in the resting or inactivated state in a concentration-dependent manner. The data indicate that the mechanisms of hKv1.5 channel inhibition by Ni²⁺ are distinct from those of other K⁺ channels. Received: 12 October 2000/Revised: 14 May 2001     

Fast inactivation in potassium channels: An interplay of cytoplasmic domains

Fast inactivation in voltage-gated potassium channels has traditionally been associated exclusively with the N-terminus. Here, we explore the role of the T1 domain using a series of chimeric channels. A chimeric channel, 4N/2, (N-terminus from the rapidly inactivating hKv1.4, and the channel body from the non-inactivating hKv1.2), exhibited slower and incomplete inactivation as compared to the wild-type hKv1.4. Replacing the T1 domain of 4N2 with that from hKv1.2 (4N/2T1/2), restored inactivation, while that from hKv1.1 (4N/1T1/2) completely abolished inactivation. Based on these observations, we hypothesize a correlation between the tetramerization domain and the putative inactivation domain receptor in the process of rapid inactivation of hKv1 channels.     

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.     

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.     

Inactivation of Kv3.3 potassium channels in heterologous expression systems

Kv3.3 K+ channels are believed to incorporate an NH2-terminal domain to produce an intermediate rate of inactivation relative to the fast inactivating K+ channels Kv3.4 and Kv1.4. The rate of Kv3.3 inactivation has, however, been difficult to establish given problems in obtaining consistent rates of inactivation in expression systems. This study characterized the properties of AptKv3.3, the teleost homologue of Kv3.3, when expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells. We show that the properties of AptKv3.3 differ significantly between CHO and HEK cells, with the largest difference occurring in the rate and voltage dependence of inactivation. While AptKv3.3 in CHO cells showed a fast and voltage-dependent rate of inactivation consistent with N-type inactivation, currents in HEK cells showed rates of inactivation that were voltage-independent and more consistent with a slower C-type inactivation. Examination of the mRNA sequence revealed that the first methionine start site had a weak Kozak consensus sequence, suggesting that the lack of inactivation in HEK cells could be due to translation at a second methionine start site downstream of the NH2-terminal coding region. Mutating the nucleotide sequence surrounding the first methionine start site to one more closely resembling a Kozak consensus sequence produced currents that inactivated with a fast and voltage-dependent rate of inactivation in both CHO and HEK cells. These results indicate that under the appropriate conditions Kv3.3 channels can exhibit fast and reliable inactivation that approaches that more typically expected of "A"-type K+ currents.     

Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels

The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.     

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.