An episodic ataxia type-1 mutation in the S1 segment sensitises the hKv1.1 potassium channel to extracellular Zn2+

Episodic ataxia type-1 (EA1) is a human neurological syndrome characterized by attacks of generalized ataxia and by continuous myokymia that has been associated with point mutations in the voltage-gated potassium channel gene KCNA1. Although important advancement has been made in understanding the molecular pathophysiology of EA1, several disease-causing mechanisms remain poorly understood. F184C is an EA1 mutation that is located within the S1 segment of the human Kv1.1 subunit. Here, we show that the F184C mutation increases approximately 4.5-fold the sensitivity of the channel to extracellular Zn2+. Both Zn2+and Cd2+ markedly alter the activation kinetics of F184C channel. In addition, the mutated channel reacts with several methane thiosulfonate reagents which specifically affected channel function. The results provide structural implications and indicate that sensitisation of hKv1.1 to Zn2+ is likely to contribute to the EA1 symptoms in patients harboring the F184C mutation.     

Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells

The choroid plexuses secrete, and maintain the composition of, the cerebrospinal fluid. K⁺ channels play an important role in these processes. In this study the molecular identity and properties of the delayed-rectifying K⁺ (Kv) conductance in rat choroid plexus epithelial cells were investigated. Whole cell K⁺ currents were significantly reduced by 10 nM dendrotoxin-K and 1 nM margatoxin, which are specific inhibitors of Kv1.1 and Kv1.3 channels, respectively. A combination of dendrotoxin-K and margatoxin caused a depolarization of the membrane potential in current-clamp experiments. Western blot analysis indicated the presence of Kv1.1 and Kv1.3 proteins in the choroid plexus. Furthermore, the Kv1.3 and Kv1.1 proteins appear to be expressed in the apical membrane of the epithelial cells in immunocytochemical studies. The Kv conductance was inhibited by 1 µM serotonin (5-HT), with maximum inhibition to 48% of control occurring in 8 min (P < 0.05 by Student's t-test for paired data). Channel inhibition by 5-HT was prevented by the 5-HT_2C antagonist mesulergine (300 nM). It was also attenuated in the presence of calphostin C (a protein kinase C inhibitor). The conductance was partially inhibited by 1,2-dioctanoyl-sn-glycerol and phorbol 12-myristate 13-acetate, both of which activate protein kinase C. These data suggest that 5-HT acts at 5-HT_2C receptors to activate protein kinase C, which inhibits the Kv channels. In conclusion, Kv1.1 and Kv1.3 channels make a significant contribution to K⁺ efflux at the apical membrane of the choroid plexus. delayed-rectifying potassium channel; serotonin     

Role of K(+) channel expression in polyamine-dependent intestinal epithelial cell migration

Polyamines are essential for cell migrationduring early mucosal restitution after wounding in the gastrointestinaltract. Activity of voltage-gated K⁺ channels (Kv) controlsmembrane potential (E_m) that regulates cytoplasmicfree Ca²⁺ concentration([Ca²⁺]_cyt) by governing thedriving force for Ca²⁺ influx. This study determinedwhether polyamines are required for the stimulation of cell migrationby altering K⁺ channel gene expression,E_m, and[Ca²⁺]_cyt in intestinal epithelialcells (IEC-6). The specific inhibitor of polyamine synthesis,-difluoromethylornithine (DFMO, 5 mM), depleted cellularpolyamines (putrescine, spermidine, and spermine), selectivelyinhibited Kv1.1 channel (a delayed-rectifier Kv channel) expression,and resulted in membrane depolarization. Because IEC-6 cells did notexpress voltage-gated Ca²⁺ channels, the depolarizedE_m in DFMO-treated cells decreased [Ca²⁺]_cyt as a result of reduceddriving force for Ca²⁺ influx through capacitativeCa²⁺ entry. Migration was reduced by 80% in thepolyamine-deficient cells. Exogenous spermidine not only reversed theeffects of DFMO on Kv1.1 channel expression, E_m,and [Ca²⁺]_cyt but also restoredcell migration to normal. Removal of extracellular Ca²⁺ orblockade of Kv channels (by 4-aminopyridine, 1-5 mM) significantly inhibited normal cell migration and prevented the restoration of cellmigration by exogenous spermidine in polyamine-deficient cells. Theseresults suggest that polyamine-dependent intestinal epithelial cellmigration may be due partially to an increase of Kv1.1 channelexpression. The subsequent membrane hyperpolarization raises[Ca²⁺]_cyt by increasing the drivingforce (the electrochemical gradient) for Ca²⁺ influx andthus stimulates cell migration.

     

Episodic ataxia type 1 mutations affect fast inactivation of K+ channels by a reduction in either subunit surface expression or affinity for inactivation domain

Episodic ataxia type 1 (EA1) is an autosomal dominant disorder characterized by continuous myokymia and episodic attacks of ataxia. Mutations in the gene KCNA1 that encodes the voltage-gated potassium channel Kv1.1 are responsible for EA1. In several brain areas, Kv1.1 coassembles with Kv1.4, which confers N-type inactivating properties to heteromeric channels. It is therefore likely that the rate of inactivation will be determined by the number of Kv1.4 inactivation particles, as set by the precise subunit stoichiometry. We propose that EA1 mutations affect the rate of N-type inactivation either by reduced subunit surface expression, giving rise to a reduced number of Kv1.1 subunits in heterotetramer Kv1.1-Kv1.4 channels, or by reduced affinity for the Kv1.4 inactivation domain. To test this hypothesis, quantified amounts of mRNA for Kv1.4 or Kv1.1 containing selected EA1 mutations either in the inner vestibule of Kv1.1 on S6 or in the transmembrane regions were injected into Xenopus laevis oocytes and the relative rates of inactivation and stoichiometry were determined. The S6 mutations, V404I and V408A, which had normal surface expression, reduced the rate of inactivation by a decreased affinity for the inactivation domain while the mutations I177N in S1 and E325D in S5, which had reduced subunit surface expression, increased the rate of N-type inactivation due to a stoichiometric increase in the number of Kv1.4 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.     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

Protein kinase C inhibits Kv1.1 potassium channel function

The regulation by protein kinase C (PKC) of recombinantvoltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC,caused persistent and large (up to 90%) inhibition of mouse, rat, andfly Shaker K currents. K currentinhibition by PMA was blocked by inhibitors of PKC, and inhibition wasnot observed in control experiments with PMA analogs that do notactivate PKC. However, site-directed substitution of potential PKCphosphorylation sites in the Kv1.1 protein did not prevent currentinhibition by PMA. Kv1.1 current inhibition was also not accompanied bychanges in macroscopic activation kinetics or in theconductance-voltage relationship. In Western blots, Kv1.1 membraneprotein was not significantly reduced by PKC activation. The injectionof oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibitionof Kv1.1 currents. These data are consistent with the hypothesis thatPKC-mediated inhibition of Kv1.1 channel function occurs by a novelmechanism that requires a C3 exoenzyme substrate but does not alterchannel activation gating or promote internalization of the channel protein.

     

Acceleration of K+ channel inactivation by MEK inhibitor U0126

Voltage-dependent (Kv)4.2-encoded A-type K⁺ channels play an important role in controlling neuronal excitability and are subject to modulation by various protein kinases, including ERK. In studies of ERK modulation, the organic compound U0126 is often used to suppress the activity of MEK, which is a kinase immediately upstream from ERK. We have observed that the inactivation time constant of heterologously expressed Kv4.2 channels was accelerated by U0126 at 1–20 µM. This effect, however, was not Kv4 family specific, because U0126 also converted noninactivating K⁺ currents mediated by Kv1.1 subunits into transient ones. To determine whether U0126 exerted these effects through kinase inhibition, we tested U0125, a derivative of U0126 that is less potent in MEK inhibition. At the same concentrations, U0125 had effects similar to those of U0126 on channel inactivation. Finally, we expressed a mutant form of Kv4.2 in which three identified ERK phosphorylation sites (T602, T607, and S616) were replaced with alanines. The inactivation of K⁺ currents mediated by this mutant was still accelerated by U0126. Our data favor the conclusion that the increase in the rate of channel inactivation by U0126 is likely to be independent of protein kinase inhibition and instead represents a direct action on channel gating. voltage-gated potassium channel; kinase; gating     

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

The possibility that protein kinase C (PKC) could control theactivity of L-type Ca²⁺ channelsin A7r5 vascular smooth muscle-derived cells in the absence of agoniststimulation was investigated using the patch-clamp technique.Consistent with the possibility that L-typeCa²⁺ channels are maximallyphosphorylated by PKC under these conditions, we show that1) activation of PKC with thephorbol ester phorbol 12,13-dibutyrate was ineffective in modulatingwhole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine orchelerythrine inhibited channel activity,3) inhibition of proteinphosphatases by intracellular dialysis of okadaic acid did not affectwhole cell currents, and 4) theinhibitory effect of staurosporine was absent in the presence ofokadaic acid. The inhibition ofCa²⁺ currents by PKC inhibitorswas due to a decrease in channel availability and long open events,whereas the voltage dependence of the open probability and thesingle-channel conductance were not affected. The evidence suggeststhat in resting, nonstimulated A7r5 cells there is a high level of PKCactivity that modulates the gating of L-typeCa²⁺ channels.

     

Episodic ataxia type-1 mutations in the Kv1.1 potassium channel display distinct folding and intracellular trafficking properties.

Episodic ataxia type 1 (EA-1) is a neurological disorder arising from mutations in the Kv1.1 potassium channel alpha-subunit. EA-1 patients exhibit substantial phenotypic variability resulting from at least 14 distinct EA-1 point mutations. We found that EA-1 missense mutations generate mutant Kv1.1 subunits with folding and intracellular trafficking properties indistinguishable from wild-type Kv1.1. However, the single identified EA-1 nonsense mutation exhibits intracellular aggregation and detergent insolubility. This phenotype can be transferred to co-assembled Kv1 alpha- and Kv beta-subunits associated with Kv1.1 in neurons. These results suggest that as in many neurodegenerative disorders, intracellular aggregation of misfolded Kv1.1-containing channels may contribute to the pathophysiology of EA-1.     

A voltage-gated K(+) current in renal inner medullary collecting duct cells

We studied the K⁺-selective conductances in primary cultures of rat renal inner medullary collecting duct (IMCD) using perforated-patch and conventional whole cell techniques. Depolarizations above –20 mV induced a time-dependent outward K⁺ current (I_vto) similar to a delayed rectifier. I_vto showed a half-maximal activation around 5.6 mV with a slope factor of 6.8 mV. Its K⁺/Na⁺ selectivity ratio was 11.7. It was inhibited by tetraethylammonium, quinidine, 4-aminopyridine, and Ba²⁺ and was not Ca²⁺ dependent. The delayed rectifying characteristics of I_vto prompted us to screen the expression of Kv1 and Kv3 families by RT-PCR. Analysis of RNA isolated from cell cultures revealed the presence of three Kv -subunits (Kv1.1, Kv1.3, and Kv1.6). Western blot analysis with Kv -subunit antibodies for Kv1.1 and Kv1.3 showed labeling of 70-kDa proteins from inner medulla plasmatic and microsome membranes. Immunocytochemical analysis of cell culture and kidney inner medulla showed that Kv1.3 is colocalized with the Na⁺-K⁺-ATPase at the basolateral membrane, although it is also in the cytoplasm. This is the first evidence of recording, protein expression, and localization of a voltage-gated Kv1 in the kidney IMCD cells. kidney; Kv1.3; potassium channel; potassium transport; whole cell clamp; immunocytochemistry; confocal microscopy     

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine

KCNQ (voltage-gated K⁺ channel family 7 (Kv7)) channels control cellular excitability and underlie the K⁺ current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K⁺ current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.     

S-acylation regulates Kv1.5 channel surface expression

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K⁺ (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH₂ and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH₂-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5. quality control; potassium; channels; palmitoylation; posttranslational     

K+ channel KV3.1 associates with OSP/claudin-11 and regulates oligodendrocyte development

K⁺ channels are differentially expressed throughout oligodendrocyte (Olg) development. K_V1 family voltage-sensitive K⁺ channels have been implicated in proliferation and migration of Olg progenitor cell (OPC) stage, and inward rectifier K+ channels (K_IR)4.1 are required for OPC differentiation to myelin-forming Olg. In this report we have identified a Shaw family K⁺ channel, K_V3.1, that is involved in proliferation and migration of OPC and axon myelination. Application of anti-K_V3.1 antibody or knockout of Kv3.1 gene decreased the sustained K⁺ current component of OPC by 50% and 75%, respectively. In functional assays block of K_V3.1-specific currents or knockout of Kv3.1 gene inhibited proliferation and migration of OPC. Adult Kv3.1 gene-knockout mice had decreased diameter of axons and decreased thickness of myelin in optic nerves compared with age-matched wild-type littermates. Additionally, K_V3.1 was identified as an associated protein of Olg-specific protein (OSP)/claudin-11 via yeast two-hybrid analysis, which was confirmed by coimmunoprecipitation and coimmunohistochemistry. In summary, the K_V3.1 K⁺ current accounts for a significant component of the total K⁺ current in cells of the Olg lineage and, in association with OSP/claudin-11, plays a significant role in OPC proliferation and migration and myelination of axons. membrane potential; tight junction; myelin; progenitor cell     

Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits.

Dendrotoxin (DTX) homologues are powerful blockers of K+ channels that contain certain subfamily Kv1 (1.1-1.6) alpha- and beta-subunits, in (alpha)4(beta)4 stoichiometry. DTXk inhibits potently Kv1.1-containing channels only, whereas alphaDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTXk with native K+ channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site-directed mutants in which amino acids were altered in the 310-helix, beta-turn, alpha-helix and random-coil regions. The mutants' antagonism of high-affinity [125I]DTXk binding to Kv1. 1-possessing channels in rat brain membranes and blockade of the Kv1. 1 current expressed in oocytes were quantified. Also, the levels of inhibition of [125I]alphaDTX binding to brain membranes by the DTXk mutants were used to measure their high- and low-affinity interactions, respectively, with neuronal Kv1.2-containing channels that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random-coil region, or R52 or R53 in the alpha-helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled alphaDTX or DTXk was dramatically reduced by alanine substitution of K26 or W25 in the beta-turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo-oligomer. The 310-helical N-terminal region of DTXk was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to approximately 1246-fold reduction in the inhibitory potency for [125I]DTXk binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTXk for Kv1.2-possessing oligomers was much less dramatic (approximately 16-fold).     

Altered gating and regulation of a carboxy-terminal ClC channel mutant expressed in the Caenorhabditis elegans oocyte

CLH-3a and CLH-3b are swelling-activated, alternatively spliced Caenorhabditis elegans ClC anion channels that have identical membrane domains but exhibit marked differences in their cytoplasmic NH₂ and COOH termini. The major differences include a 71-amino acid CLH-3a NH₂-terminal extension and a 270-amino acid extension of the CLH-3b COOH terminus. Splice variation gives rise to channels with striking differences in voltage, pH, and Cl^– sensitivity. On the basis of structural and functional insights gained from crystal structures of bacterial ClCs, we suggested previously that these functional differences are due to alternative splicing of the COOH terminus that may change the accessibility and/or function of pore-associated ion-binding sites. We recently identified a mutant worm strain harboring a COOH-terminal deletion mutation in the clh-3 gene. This mutation removes 101 COOH-terminal amino acids unique to CLH-3b and an additional 64 upstream amino acids shared by both channels. CLH-3b is expressed in the worm oocyte, which allowed us to characterize the mutant channel, CLH-3bC, in its native cellular environment. CLH-3bC exhibits altered voltage-dependent gating as well as pH and Cl^– sensitivity that resemble those of CLH-3a. This mutation also alters channel inhibition by Zn²⁺, prevents ATP depletion-induced activation, and dramatically reduces volume sensitivity. These results suggest that the deleted COOH-terminal region of CLH-3bC functions to modulate channel sensitivity to voltage and extracellular ions. This region also likely plays a role in channel regulation and cell volume sensitivity. Our findings contribute to a growing body of evidence indicating that cytoplasmic domains play key roles in the gating and regulation of eukaryotic ClCs. chloride; cell volume; voltage-gated anion channel     

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvbeta1.1 subunits modulated by G protein beta gamma subunits

Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.     

Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents

Cell shrinkageis an early prerequisite in programmed cell death, and cytoplasmicK⁺ is a dominant cation that controls intracellular ionhomeostasis and cell volume. Blockade of K⁺ channelsinhibits apoptotic cell shrinkage and attenuates apoptosis. We examined whether apoptotic repressor with caspase recruitment domain (ARC), an antiapoptotic protein, inhibits cardiomyocyte apoptosis by reducing K⁺ efflux throughvoltage-gated K⁺ (Kv) channels. In heart-derived H9c2cells, whole cell Kv currents (I_K(V)) wereisolated by using Ca²⁺-free extracellular (bath) solutionand including 5 mM ATP and 10 mM EGTA in the intracellular (pipette)solution. Extracellular application of 5 mM 4-aminopyridine (4-AP), ablocker of Kv channels, reversibly reduced I_K(V)by 50-60% in H9c2 cells. The remaining currents during 4-APtreatment may be generated by K⁺ efflux through4-AP-insensitive K⁺ channels. Overexpression of ARC inheart-derived H9c2 cells significantly decreasedI_K(V), whereas treatment with staurosporine, apotent apoptosis inducer, enhanced I_K(V)in wild-type cells. The staurosporine-induced increase inI_K(V) was significantly suppressed and thestaurosporine-mediated apoptosis was markedly inhibited incells overexpressing ARC compared with cells transfected with thecontrol neomycin vector. These results suggest that theantiapoptotic effect of ARC is, in part, due to inhibition of Kvchannels in cardiomyocytes.

     

A Kv channel with an altered activation gate sequence displays both "fast" and "slow" activation kinetics

The Kv1–4 families of K⁺ channels contain a tandem proline motif (PXP) in the S6 helix that is crucial for channel gating. In human Kv1.5, replacing the first proline by an alanine resulted in a nonfunctional channel. This mutant was rescued by introducing another proline at a nearby position, changing the sequence into AVPP. This resulted in a channel that activated quickly (ms range) upon the first depolarization. However, thereafter, the channel became trapped in another gating mode that was characterized by slow activation kinetics (s range) with a shallow voltage dependence. The switch in gating mode was observed even with very short depolarization steps, but recovery to the initial "fast" mode was extremely slow. Computational modeling suggested that switching occurred during channel deactivation. To test the effect of the altered PXP sequence on the mobility of the S6 helix, we used molecular dynamics simulations of the isolated S6 domain of wild type (WT) and mutants starting from either a closed or open conformation. The WT S6 helix displayed movements around the PXP region with simulations starting from either state. However, the S6 with a AVPP sequence displayed flexibility only when started from the closed conformation and was rigid when started from the open state. These results indicate that the region around the PXP motif may serve as a "hinge" and that changing the sequence to AVPP results in channels that deactivate to a state with an alternate configuration that renders them "reluctant" to open subsequently. voltage-gated potassium channel     

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. potassium channel inactivation; potassium channel ancillary subunits; closed-state inactivation; voltage-gated potassium channels