Closed-state Inactivation Of Kv4 Research Articles

Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation

We studied the kinetics and structural determinants of closed-state inactivation (CSI) in Kv4.2 channels, considering a multistep process and the possibility that both intra- and intersubunit dynamic binding (i.e., loss and restoration of physical contact) may occur between the S4-S5 linker, including the initial S5 segment (S4S5), and the S6 gate. We expressed Kv4.2 channels in Xenopus oocytes and measured the onset of low-voltage inactivation under two-electrode voltage clamp. Indicative of a transitory state, the onset kinetics were best described by a double-exponential function. To examine the involvement of individual S4S5 and S6 amino acid residues in dynamic binding, we studied S4S5 and S6 single alanine mutants and corresponding double mutants. Both transitory and steady-state inactivation were modified by these mutations, and we quantified the mutational effects based on apparent affinities for the respective inactivated states. Double-mutant cycle analyses revealed strong functional coupling of the S6 residues V404 and I412 to all tested S4S5 residues. To examine whether dynamic S4S5/S6 binding occurs within individual α-subunits or between neighboring α-subunits, we performed a double-mutant cycle analysis with Kv4.2 tandem-dimer constructs. The constructs carried either an S4S5/S6 double mutation in the first α-subunit and no mutation in the second (concatenated) α-subunit or an S4S5 point mutation in the first α-subunit and an S6 point mutation in the second α-subunit. Our results support the notion that CSI in Kv4.2 channels is a multistep process that involves dynamic binding both within individual α-subunits and between neighboring α-subunits.

Effect of mosapride on Kv4.3 potassium channels expressed in CHO cells

Mosapride and cisapride are gastroprokinetic agents with 5-hydroxytryptamine4 receptor agonist activity and have been widely used in the treatment of a variety of gastrointestinal disorders. The effects of mosapride and cisapride on cloned Kv4.3 channels stably expressed in Chinese hamster ovary cells were investigated using the whole-cell patch-clamp technique. Mosapride and cisapride inhibited Kv4.3 in a concentration-dependent manner with IC50 values of 15.2 and 9.8μM, respectively. Mosapride accelerated the rate of inactivation and activation of Kv4.3 in a concentration-dependent manner and thereby decreased the time to peak. The rate constants of association (k +1) and dissociation (k -1) for mosapride were 9.9μM(-1)s(-1) and 151.3s(-1), respectively. The K D (k -1/k +1) was 16.2μM, similar to the IC50 value calculated from the concentration-response curve. Voltage-dependent inhibition by mosapride was observed in the voltage range for channel opening but was not observed over a voltage range in which all Kv4.3 channels were open. Both the steady-state activation and inactivation curves of Kv4.3 were shifted in the hyperpolarizing direction in the presence of mosapride. Mosapride also caused a substantial acceleration in closed-state inactivation of Kv4.3. Mosapride produced use-dependent inhibition, which was consistent with a slow recovery from inactivation of Kv4.3. M1 and norcisapride, the major metabolites of mosapride and cisapride, respectively, had little or no effect on Kv4.3. These results indicate that mosapride inhibits Kv4.3 by both preferential binding to the open state of the channels during depolarization and acceleration of the closed-state inactivation at subthreshold potentials.

Inhibition of Kv4.3 potassium channels by trazodone

Trazodone, a triazolopyridine antidepressant, is commonly used in the treatment of depression and insomnia. Kv4.3 channels are transiently, and rapidly, inactivating Kv channels that are highly expressed in cardiac myocytes and neurons. To determine the electrophysiological basis for the cardiac and neuronal actions of trazodone, we studied the effects of trazodone on Kv4.3 currents stably expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Trazodone decreased the peak amplitude of Kv4.3 in a concentration-dependent manner with an IC50 of 55.4μM. Under control conditions, the time course of inactivation of Kv4.3 at +40mV was fitted to a double exponential function. Trazodone produced a concentration-dependent slowing of the fast and slow components of Kv4.3 inactivation during a voltage step to +40mV. The inhibition of Kv4.3 by trazodone was voltage independent over the entire voltage range tested. Trazodone shifted the voltage dependence of the steady-state inactivation of Kv4.3 to a hyperpolarizing direction. However, the slope factor of the steady-state inactivation was not affected by trazodone. Under control conditions, the closed-state inactivation of Kv4.3 was fitted to a single exponential function. Trazodone significantly accelerated the closed-state inactivation of Kv4.3. Trazodone produced a weak use-dependent inhibition of Kv4.3 at frequencies of 1 and 2Hz. m-Chlorophenylpiperazine (m-CPP), a major metabolite of trazodone, inhibited Kv4.3 less potently than trazodone, with an IC50 of 118.6μM. These results suggest that trazodone preferentially inhibited Kv4.3 by both binding to the closed state and accelerating the closed-state inactivation of the channel.

External Ba2+ block of Kv4.2 channels is enhanced in the closed-inactivated state

The effect of external barium ions on rat Kv4.2 channels expressed in HEK293 cells was investigated using whole cell, voltage-clamp recordings to determine its mechanism of action as well as its usefulness as a tool to probe the permeation pathway. Ba(2+) caused a concentration-dependent inhibition of current that was antagonized by increasing the external concentration of K(+) ([K(+)](o)), and the concentration and time dependence of the inhibition were well fitted by a model involving two binding sites aligned in series. Recovery from current inhibition was enhanced by increasing the intensity, duration, or frequency of depolarizing steps or by increasing [K(+)](o). These properties are consistent with the conclusion that Ba(2+) is a permeant ion that, by virtue of a stable interaction with a deep pore site, is able to block conduction. This blocking action was subsequently exploited to gain insights into the pore configuration in different channel states. In addition to blocking one or more states populated by brief depolarizing pulses to 80 mV, Ba(2+) blocked closed channels [the membrane voltage (V(m)) = -80 mV] and closed-inactivated channels (V(m) = -40 mV). Interestingly, the block of closed-inactivated channels was faster and more complete than for closed channels, which we interpret to mean that conformational changes underlying closed-state inactivation (CSI) enhance Ba(2+) binding and that the outer pore mouth remains patent during CSI. This provides the first direct evidence that an inactivation process involving a constriction of the outer pore mouth does not account for CSI in Kv4.2.

A Limited 4 Å Radial Displacement of the S4-S5 Linker Is Sufficient for Internal Gate Closing in Kv Channels

Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3-4 Å is sufficient to prevent ion permeation through the pore.

Enhancement of Closed-State Inactivation and ER Retention of Kv4.3 Mediated by N-Terminal KIS Domain of Auxiliary KChIP4A

Auxiliary KChIP4a shares high homology of conserved C-terminal core region with other members of Kv channel-interacting proteins (KChIPs), but exhibits a distinct modulation on Kv4 current expression and gating. It has been shown that the unique N-terminus of KChIP4a functions as K+ channel inactivation suppressor (KIS) that leads to slow inactivation and current inhibition. However, the mechanism by which the KIS domain causes current reduction remains unknown. In this study, we identified a hydrophobic ER-retention motif within the KIS domain of KChIP4a that suppresses Kv4.3 surface expression using confocal imaging and cell surface biotinylation assay. Further dissection of KIS domain revealed several key residues that cause reduction of Kv4.3 peak current, but do not affect surface expression. Examination of gating properties of Kv4.3 co-expressed with either KChIP4a or its core without the KIS (KChIP4aΔ2-34) demonstrated that KChIP4a with KIS domain had no significant effect on steady-state activation, but shifted the voltage dependence of steady-state inactivation of Kv4.3 to hyperpolarizing direction by enhancing closed-state inactivation. We have previously demonstrated that KChIP4a can rescue the function of a tetramerization-defect mutant Kv4.3 C110A. The rescued Kv4.3 C110A current is larger than that of WT Kv4.3/KChIP4a co-expression, although C110A surface expression was lower. Upon coexpression, the closed-state inactivation of Kv4.3 C110A mutant was less affected by the KIS domain, as compared with enhanced closed-state inactivation observed in wild-type channel, suggesting a role of T1 domain in mediation of Kv4 closed-state inactivation. Taken together, we propose that N-terminal KIS domain of KChIP4a inhibits Kv4.3 function through dual independent mechanisms by which auxiliary KChIP4a causes Kv4 ER retention and promotes channel closed-state inactivation.

Closed-State Inactivation of a Voltage-Gated Potassium Channel Involves Inter-Subunit Interactions

We study the structural determinants of inactivation in voltage-gated potassium (Kv) channels of the Kv4 subfamily, which exhibit preferential closed-state inactivation (CSI). There is strong evidence that dynamic coupling between the S4S5 linker (S4S5) and the main S6 activation gate (S6) plays a central role in CSI (Barghaan and Bähring, J Gen Physiol 133: 205-224, 2009). In particular, absence or loss of contact between S4S5 and S6 is thought to uncouple the activation gate from the voltage sensor. Here we examined the role of dynamic coupling between the S4S5 residue Phe 326 and the S6 residue Val 404 in CSI. We expressed Kv4.2 wild-type (wt), the single mutants Kv4.2 F326A and Kv4.2 V404A, and the double mutant Kv4.2 F326A:V404A in Xenopus oocytes and measured CSI parameters of these constructs under two-electrode voltage-clamp. Double-mutant cycle analysis revealed strong thermodynamic coupling between Phe 326 in S4S5 and Val 404 in S6 during CSI (ln Omega at −65 mV = 1.85). Next we asked whether this dynamic coupling occurs between S4S5 and S6 of individual alpha-subunits (loss of intra-subunit contact) or two neighboring alpha-subunits (loss of inter-subunit contact). To answer this question we created dimer constructs of Kv4.2 (1) with a point mutation in S6 of one subunit and a point mutation in S4S5 of the second subunit (Kv4.2 V404A_F326A), and (2) with a double point mutation in one subunit and no mutation in the second subunit (Kv4.2 F326A:V404A_wt). Notably, only the dimer Kv4.2 V404A_F326A was able to reproduce the CSI properties observed when Kv4.2 F326A:V404A monomers were expressed, whereas the dimer Kv4.2 F326A:V404A_wt resembled wild-type. Our data support the notion that CSI in Kv4.2 channels involves dynamic coupling between neighboring subunits. Supported by the Deutsche Forschungsgemeinschaft (DFG).

Ancillary Subunits Regulate PKC Mediated Effects on Closed-State Inactivation of Kv4.3

Kv4 channels are expressed with a variety of ancillary subunits in vivo. The most prominent of these proteins are the KChIPs, a family of cytoplasmic proteins that modulate channel gating and act as chaperones. We have previously shown that heterologously expressed Kv4.3 is regulated by PKC. After induction of PKC by PMA, current expression level is reduced. PKC also influences closed-state inactivation (CSI) in an isoform dependent manner; CSI is decreased upon PKC induction in Kv4.3-S, and increased in PKC-L. To understand the role of PKC on modulation of Kv4-based currents, we expressed channels in the presence of three KChIP2 isoforms and compared their responses to PKC. Two KChIP2 isoforms, KChIP2a and 2b, negated PKC influence on channel gating, kinetics, and expression levels in both Kv4.3-S and Kv4.3-L. In contrast, 70-amino acid KChIP2d was permissive for PKC modulation with respect to both CSI and current expression, while allowing the fast recovery from open-state inactivation characteristic of other KChIP2 isoforms. Additionally, the KChIP2d effects on CSI in Kv4.3-L were dependent on the presence of a putative PKC phosphorylation site in the C terminus. These data suggest a different physiological role for KChIP2d than the other KChIP2 isoforms, and suggest that the longer forms of KChIP2 interact with the regions of Kv4.3 affected by PKC, while KChIP2d interacts with the channel in a manner that allows PKC modulation while still accelerating recovery.

Effects of PKC on Closed-State Inactivation in Kv4.3 Isoforms

Kv4.3 is expressed as two isoforms, a short form and a long form, that has a 19 aa insertion downstream from S6 in the C terminus which has a putative PKC phosphorylation site at T504. To understand the role of PKC on modulation of closed-state inactivation (CSI), we expressed channels mutated in putative PKC phosphorylation sites and compared their response to PKC activation with PMA to the responses of WT Kv4.3 isoforms. PMA had similar effects on Kv4.3-S and Kv4.3-L open-state inactivation. However, PMA induced opposite effects on CSI in the two channel splice variants: the magnitude of CSI in Kv4.3-S was reduced, while there was an increase in CSI in Kv4.3-L, an effect was abolished by mutation of the long form T504 to alanine. To understand the structural basis of the reduction of CSI in Kv4.3-S, we constructed several mutants of putative PKC phosphorylation sites in the N terminus. Of these, the largest effect on PKC modulation of CSI occurred in the T53A mutant in both Kv4.3-S and Kv4.3-L; Kv4.3-S mutants lacking threonine at position 53 showed no or minimal response to PKC. These data show that isoform-specific modulation of CSI by PKC in Kv4.3 involves complex interactions of the cytoplasmic N and C termini of the channels.

Closed-state inactivation in Kv4.3 isoforms is differentially modulated by protein kinase C

Kv4.3, with its complex open- and closed-state inactivation (CSI) characteristics, is a primary contributor to early cardiac repolarization. The two alternatively spliced forms, Kv4.3-short (Kv4.3-S) and Kv4.3-long (Kv4.3-L), differ by the presence of a 19-amino acid insert downstream from the sixth transmembrane segment. The isoforms are similar kinetically; however, the longer form has a unique PKC phosphorylation site. To test the possibility that inactivation is differentially regulated by phosphorylation, we expressed the Kv4.3 isoforms in Xenopus oocytes and examined changes in their inactivation properties after stimulation of PKC activity. Whereas there was no difference in open-state inactivation, there were profound differences in CSI. In Kv4.3-S, PMA reduced the magnitude of CSI by 24% after 14.4 s at -50 mV. In contrast, the magnitude of CSI in Kv4.3-L increased by 25% under the same conditions. Mutation of a putatively phosphorylated threonine (T504) to aspartic acid within a PKC consensus recognition sequence unique to Kv4.3-L eliminated the PMA response. The change in CSI was independent of the intervention used to increase PKC activity; identical results were obtained with either PMA or injected purified PKC. Our previously published 11-state model closely simulated our experimental data. Our data demonstrate isoform-specific regulation of CSI by PKC in Kv4.3 and show that the carboxy terminus of Kv4.3 plays an important role in regulation of CSI.

Closed-State Inactivation in Kv4.3 Splice Forms is Differentially Modulated by Protein Kinase C

Kv4.3, with its complex open- and closed-state inactivation (CSI) characteristics, is a primary contributor to early cardiac repolarization. The two alternatively-spliced forms of Kv4.3 (L and S) differ by the presence of a 19 amino acid exon 81 amino acids downstream from the sixth transmembrane segment. The two isoforms are reported to be kinetically similar, however, the longer form has a unique PKC phosphorylation site. To test the possibility that inactivation is differentially regulated by phosphorylation, we stimulated PKC in Xenopus oocytes expressing Kv4.3 isoforms and examined their inactivation properties. There was no difference in open-state inactivation, there were profound differences in CSI; in Kv4.3-S, PMA (10nM) reduced the extent of CSI from 0.53±0.06 to 0.69±0.05 after 14.4 s at −50 mV. In contrast, CSI in Kv4.3-L increased from 0.71±0.05 to 0.51±0.03 under the same conditions. Mutation of the unique putative PKC phosphorylation site in Kv4.3-L eliminated its isoform-specific behavior. This effect was independent of the intervention used to increase PKC activity; identical results were obtained with either PMA or injected purified PKC. Our data demonstrate that Kv4.3 can be differentially regulated by PKC, and that the carboxy terminus of Kv4.3 plays an important role in regulation of CSI.

Dynamic Coupling of Voltage Sensor and Gate Involved in Closed-State Inactivation of Kv4.2 Channels

Voltage-gated potassium channels related to the Shal-gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (ISA) which controls dendritic excitation and the backpropagation of action potentials in neurons. Kv4 channels also exhibit a prominent low-voltage-induced closed-state inactivation, but the underlying mechanism is poorly understood. We examined a structural model, in which uncoupling of the voltage sensor from the cytoplasmic gate mediates inactivation in Kv4.2 channels. Support for such a model comes from our finding that chimeric swapping of S4-S5 linker and distal S6 sequences between N-terminally truncated Kv4.2Δ2-40 and ShakerIR channels slowed inactivation of the former and induced a form of fast macroscopic inactivation in the latter under two-electrode voltage-clamp in Xenopus oocytes. We performed a Kv4.2 alanine scanning-mutagenesis in the S4-S5 linker, the initial part of S5, and the distal part of S6 and functionally characterized these mutants. In a large fraction of the mutants (> 80%) normal channel function was preserved, but the mutations influenced the likelihood of the channel to enter the closed-inactivated state. Depending on the site of mutation, the onset kinetics of low-voltage inactivation and/or the kinetics of recovery from inactivation were accelerated or slowed and the voltage dependence of steady-state inactivation was shifted positive or negative. In some mutants these inactivation parameters remained unaffected. Double-mutant cycle analysis based on kinetic and steady-state parameters of low-voltage inactivation revealed that residues known to be critical for voltage-dependent gate-opening, including Glu 323 and Val 404, are also critical for Kv4.2 closed-state inactivation. Selective redox modulation of corresponding double-cysteine mutants by dithiothreitol (DTT) tert-butyl hydroperoxide (tbHO2) supported the idea that these residues are involved in a dynamic coupling, which mediates both transient activation and closed-state inactivation in Kv4.2 channels.

Dynamic Coupling of Voltage Sensor and Gate Involved in Closed-State Inactivation of Kv4.2 Channels

Voltage-gated potassium channels related to the Shal gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (ISA) that controls dendritic excitation and the backpropagation of action potentials in neurons. Kv4 channels also exhibit a prominent low voltage–induced closed-state inactivation, but the underlying molecular mechanism is poorly understood. Here, we examined a structural model in which dynamic coupling between the voltage sensors and the cytoplasmic gate underlies inactivation in Kv4.2 channels. We performed an alanine-scanning mutagenesis in the S4-S5 linker, the initial part of S5, and the distal part of S6 and functionally characterized the mutants under two-electrode voltage clamp in Xenopus oocytes. In a large fraction of the mutants (>80%) normal channel function was preserved, but the mutations influenced the likelihood of the channel to enter the closed-inactivated state. Depending on the site of mutation, low-voltage inactivation kinetics were slowed or accelerated, and the voltage dependence of steady-state inactivation was shifted positive or negative. Still, in some mutants these inactivation parameters remained unaffected. Double mutant cycle analysis based on kinetic and steady-state parameters of low-voltage inactivation revealed that residues known to be critical for voltage-dependent gate opening, including Glu 323 and Val 404, are also critical for Kv4.2 closed-state inactivation. Selective redox modulation of corresponding double-cysteine mutants supported the idea that these residues are involved in a dynamic coupling, which mediates both transient activation and closed-state inactivation in Kv4.2 channels.

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4

Voltage-gated K(+) channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K(+) currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At -60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K(+) channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K(+) channels.