Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.     

Structural determinants of Kvbeta1.3-induced channel inactivation: a hairpin modulated by PIP2

Inactivation of voltage-gated Kv1 channels can be altered by Kvbeta subunits, which block the ion-conducting pore to induce a rapid ('N-type') inactivation. Here, we investigate the mechanisms and structural basis of Kvbeta1.3 interaction with the pore domain of Kv1.5 channels. Inactivation induced by Kvbeta1.3 was antagonized by intracellular PIP(2). Mutations of R5 or T6 in Kvbeta1.3 enhanced Kv1.5 inactivation and markedly reduced the effects of PIP(2). R5C or T6C Kvbeta1.3 also exhibited diminished binding of PIP(2) compared with wild-type channels in an in vitro lipid-binding assay. Further, scanning mutagenesis of the N terminus of Kvbeta1.3 revealed that mutations of L2 and A3 eliminated N-type inactivation. Double-mutant cycle analysis indicates that R5 interacts with A501 and T480 of Kv1.5, residues located deep within the pore of the channel. These interactions indicate that Kvbeta1.3, in contrast to Kvbeta1.1, assumes a hairpin structure to inactivate Kv1 channels. Taken together, our findings indicate that inactivation of Kv1.5 is mediated by an equilibrium binding of the N terminus of Kvbeta1.3 between phosphoinositides (PIPs) and the inner pore region of the channel.     

Differential modulation of Kv1 channel-mediated currents by co-expression of Kvbeta3 subunit in a mammalian cell-line

The effect of Kvbeta3 subunit co-expression on currents mediated by the Shaker-related channels Kv1.1 to Kv1.6 in Chinese hamster ovary (CHO) cells was studied with patch-clamp techniques. In the presence of Kvbeta3, differences in the voltage dependence of activation for Kv1.1, Kv1.3 and Kv1.6 were detected, but not for Kv1.2- and Kv1.4-mediated currents. Co-expression of Kvbeta3 did not cause a significant increase in current density for any of the tested channels. In contrast to previous studies in Xenopus oocyte expression system, Kvbeta3 confered a rapid inactivation to all except Kv1.3 channels. Also, Kv1.6 channels that possess an N-type inactivation prevention (NIP) domain for Kvbeta1.1, inactivated rapidly when co-expressed with Kvbeta3. Onset and recovery kinetics of channel inactivation distinctly differed for the various Kv1alpha/Kvbeta3 subunit combinations investigated in this study. The results indicate that the choice of expression system may critically determine Kvbeta3 inactivating activity. This suggests that the presence of an inactivating domain and a receptor in a channel pore, although necessary, may not be sufficient for an effective rapid N-type inactivation of Kv1 channels in heterologous expression systems.     

Protein kinase A phosphorylation alters Kvbeta1.3 subunit-mediated inactivation of the Kv1.5 potassium channel

The human Kv1.5 potassium channel forms the IKur current in atrial myocytes and is functionally altered by coexpression with Kvbeta subunits. To explore the role of protein kinase A (PKA) phosphorylation in beta-subunit function, we examined the effect of PKA stimulation on Kv1.5 current following coexpression with either Kvbeta1.2 or Kvbeta1.3, both of which coassemble with Kv1.5 and induce fast inactivation. In Xenopus oocytes expressing Kv1.5 and Kvbeta1.3, activation of PKA reduced macroscopic inactivation with an increase in K+ current. Similar results were obtained using HEK 293 cells which lack endogenous K+ channel subunits. These effects did not occur when Kv1.5 was coexpressed with either Kvbeta1.2 or Kvbeta1.3 lacking the amino terminus, suggesting involvement of this region of Kvbeta1.3. Removal of a consensus PKA phosphorylation site on the Kvbeta1.3 NH2 terminus (serine 24), but not alternative sites in either Kvbeta1.3 or Kv1.5, resulted in loss of the functional effects of kinase activation. The effects of phosphorylation appeared to be electrostatic, as replacement of serine 24 with a negatively charged amino acid reduced beta-mediated inactivation, while substitution with a positively charged residue enhanced it. These results indicate that Kvbeta1.3-induced inactivation is reduced by PKA activation, and that phosphorylation of serine 24 in the subunit NH2 terminus is responsible.     

Anorexic effect of K+ channel blockade in mesenteric arterial smooth muscle and intestinal epithelial cells.

Activity of voltage-gated K+ (Kv) channels controls membrane potential (E(m)). Membrane depolarization due to blockade of K+ channels in mesenteric artery smooth muscle cells (MASMC) should increase cytoplasmic free Ca2+ concentration ([Ca2+]cyt) and cause vasoconstriction, which may subsequently reduce the mesenteric blood flow and inhibit the transportation of absorbed nutrients to the liver and adipose tissue. In this study, we characterized and compared the electrophysiological properties and molecular identities of Kv channels and examined the role of Kv channel function in regulating E(m) in MASMC and intestinal epithelial cells (IEC). MASMC and IEC functionally expressed multiple Kv channel alpha- and beta-subunits (Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.5, Kv2.1, Kv4.3, and Kv9.3, as well as Kvbeta1.1, Kvbeta2.1, and Kvbeta3), but only MASMC expressed voltage-dependent Ca2+ channels. The current density and the activation and inactivation kinetics of whole cell Kv currents were similar in MASMC and IEC. Extracellular application of 4-aminopyridine (4-AP), a Kv-channel blocker, reduced whole cell Kv currents and caused E(m) depolarization in both MASMC and IEC. The 4-AP-induced E(m) depolarization increased [Ca2+]cyt in MASMC and caused mesenteric vasoconstriction. Furthermore, ingestion of 4-AP significantly reduced the weight gain in rats. These results suggest that MASMC and IEC express multiple Kv channel alpha- and beta-subunits. The function of these Kv channels plays an important role in controlling E(m). The membrane depolarization-mediated increase in [Ca2+]cyt in MASMC and mesenteric vasoconstriction may inhibit transportation of absorbed nutrients via mesenteric circulation and limit weight gain.     

Coupling of voltage-dependent potassium channel inactivation and oxidoreductase active site of Kvbeta subunits

The accessory beta subunits of voltage-dependent potassium (Kv) channels form tetramers arranged with 4-fold rotational symmetry like the membrane-integral and pore-forming alpha subunits (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell. 90, 943-952). The crystal structure of the Kvbeta2 subunit shows that Kvbeta subunits are oxidoreductase enzymes containing an active site composed of conserved catalytic residues, a nicotinamide (NADPH)-cofactor, and a substrate binding site. Also, Kvbeta subunits with an N-terminal inactivating domain like Kvbeta1.1 (Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, O., and Pongs, O. (1994) Nature 369, 289-294) and Kvbeta3.1 (Heinemann, S. H., Rettig, J., Graack, H. R., and Pongs, O. (1996) J. Physiol. (Lond.) 493, 625-633) confer rapid N-type inactivation to otherwise non-inactivating channels. Here we show by a combination of structural modeling and electrophysiological characterization of structure-based mutations that changes in Kvbeta oxidoreductase activity may markedly influence the gating mode of Kv channels. Amino acid substitutions of the putative catalytic residues in the Kvbeta1.1 oxidoreductase active site attenuate the inactivating activity of Kvbeta1.1 in Xenopus oocytes. Conversely, mutating the substrate binding domain and/or the cofactor binding domain rescues the failure of Kvbeta3.1 to confer rapid inactivation to Kv1.5 channels in Xenopus oocytes. We propose that Kvbeta oxidoreductase activity couples Kv channel inactivation to cellular redox regulation.     

Influence of permeating ions on Kv1.5 channel block by nifedipine

Nifedipine can block K(+) currents through Kv1.5 channels in an open-channel manner (32). Replacement of internal and external K(+) with equimolar Rb(+) or Cs(+) reduced the potency of nifedipine block of Kv1.5 from an IC(50) of 7.3 microM (K(+)) to 16.0 microM (Rb(+)) and 26.9 microM (Cs(+)). The voltage dependence of block was unaffected, and a single binding site block model was used to describe block for all three ions. By varying ion species at the intra- and extracellular mouth of the channel and by using a nonconducting W472F-Kv1.5 mutant, we demonstrated that block was conditioned by the ion permeating the pore and, to a lesser extent, by the extracellular ion species alone. In Kv1.5, the outer pore mutations R487V and R487Y reduced nifedipine potency close to that of Kv4.2 and other Kv channels with an equivalent valine. Although changing this residue can affect C-type inactivation of Kv channels, the normalized reduction and time course of currents blocked by nifedipine in 5, 135, and 300 mM extracellular K(+) concentration was the same. Similarly, a mean recovery time constant from nifedipine block of 316 ms was unchanged (332 ms) after 5-s prepulses to allow C-type inactivation. This is consistent with the conclusion that nifedipine block and C-type inactivation in the Kv1.5 channel can coexist but are mediated by distinct mechanisms coordinated by outer pore conformation.     

KChAP/Kvbeta1.2 interactions and their effects on cardiac Kv channel expression

KChAP and voltage-dependent K+ (Kv) beta-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta-subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta1.2, and Kvbeta1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.     

Disinactivation of N-type inactivation of voltage-gated K channels by an erbstatin analogue

In some A-type voltage-gated K channels, rapid inactivation is achieved through the binding of an N-terminal domain of the pore-forming alpha-subunit or an associated beta-subunit to a cytoplasmic acceptor located at or near the channel pore using the ball-and-chain machinery (1-5). This inactivation involving the N terminus is known as N-type inactivation. Here, we describe an erbstatin (Erb) analogue as a small molecule inhibitor of the N-type inactivation in channels of Kv1.4 and Kv1.1+Kvbeta1. We show that this inhibition of inactivation (designated as "disinactivation") is potent and selective for N-type inactivation in heterologous cells (Chinese hamster ovary and Xenopus oocytes) expressing these A-type channels. In Chinese hamster ovary cells, Erb increased the inactivation time constant of Kv1.4 from 86.5 +/- 9.5 to 150 +/- 10 ms (n = 6, p < 0.0 1). Similarly, Erb increased the inactivation time constant of Kv1.1+Kvbeta1 from 10 +/- 0.9 to 49.3 +/- 7 ms (n = 7, p < 0.01). The EC(50) for disinactivating Kv1.1+Kvbeta1 was 10.4 +/- 0.9 microm (n = 2-9). Erb had no effect upon another A-channel, Kv4.3, which does not utilize the ball-and-chain mechanism. The mechanism of Erb-induced disinactivation was also investigated. Neither cysteine oxidation nor tyrosine kinase inhibition was involved. The results demonstrate that Erb can be used as a base structure to identify potent, selective small molecule inhibitors of intracellular protein-protein interactions, and that these disinactivators may offer another therapeutic approach to the treatment of seizure disorders.     

Disruption of Kv1.1 N-type inactivation by novel small molecule inhibitors (disinactivators)

Kv1.1 channels are expressed in many regions of the brain and spinal cord [Monaghan, M. M.; Trimmer, J. S.; Rhodes, K. J. J. Neurosci.2001, 21, 5973; Rasband, M. N.; Trimmer, J. S. J. Comp. Neurol.2001, 429, 166; Trimmer, J. S.; Rhodes, K. J. Ann. Rev. Physiol.2004, 66, 477]. When expressed alone, they produce a delayed rectifier slowly inactivating type current that contributes to hyperpolarizing the neuron following depolarization. In the hippocampus Kv1.1 is co-expressed with Kvbeta1 (and other beta subunits), which converts Kv1.1 into a transient, fast inactivating current, reducing its ability to hyperpolarize the cell and thus increasing neuronal excitability. To reduce neuronal excitability, screening for compounds that prevent inactivation of Kv1.1 channels by Kvbeta1 was performed using a yeast two-hybrid screen. A variety of compounds were discovered in this assay and subsequently determined to disrupt inactivation of the ionic currents, and hence were termed 'disinactivators'. Several of these disinactivators also inhibited pentylenetetrazole-induced seizures (PTZ) in mice. Compounds were found to act by several mechanisms to prevent Kvbeta1 inactivation of Kv1.1 channels, including enhancement of Ca(2+) release/influx and by direct mechanisms. Two structural classes were identified that act on a Kvbeta1N70-Kv1.1 chimera where the N-terminal 70 amino acids of Kvbeta1 were attached to the N-terminus of Kv1.1. It is likely that these disinactivators act directly on the Kvbeta1 N-terminus or its receptor site on Kv1.1, thus preventing it from blocking Kv1.1 channels. Compounds acting by this mechanism may be useful for reducing neuronal hyperexcitability in diseases such as epilepsy and neuropathic pain.     

Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4

Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents. The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme (). To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 in either the NADPH docking or putative catalytic sites. Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kv beta 2 with Kv alpha 1 channels. To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes. Kv beta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents. The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased. Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current. Western blots showed that both types of mutation reduced Kv1.4 protein. Like the wild-type Kv beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4. Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 on Kv1.4 currents from its effect on the rate of inactivation. These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kv beta 2 may play a role in the processing of Kv1.4.     

The inhibitory effect of copper ions on lymphocyte Kv1.3 potassium channels.

We applied the whole-cell patch-clamp technique to study the inhibitory effect of copper ions (Cu) on the activity of Kv1.3 channels expressed in human lymphocytes. Application of Cu reversibly inhibited the currents to about 10% of the control value in a concentration-dependent manner with the half blocking concentration of 5.28+/-0.5 microM and the Hill's coefficient of 3.83+/-0.18. The inhibitory effect was saturated at 10 microM concentration. The inhibition was time-dependent and it was correlated in time with a significant slowing of the current activation rate. In contrast the voltage dependence of activation was not changed by Cu as well as the inactivation kinetics. The inhibitory effect of Cu was voltage-independent. It was also unaffected by changing the extracellular pH in the range from 6.4 to 8.4, raising the extracellular potassium concentration to 150 mM and by changing the holding potential from -90 to -60 mV. The inhibitory effect of Cu was not changed in the presence of an equivalent concentration of Zn. Altogether, obtained data suggest that Cu inhibits Kv1.3 channels by a different mechanism than Zn and that Cu and Zn act on different binding sites. The inhibitory effect of Cu was probably due to a specific binding of Cu on binding sites on the channels. Possible physiological significance of the Cu-induced inhibition of Kv1.3 channels is discussed.     

Functional coupling between the Kv1.1 channel and aldoketoreductase Kvbeta1

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Effects of changes in extracellular pH and potassium concentration on Kv1.3 inactivation

The Kv1.3 channel inactivates via the P/C-type mechanism, which is influenced by a histidine residue in the pore region (H399, equivalent of Shaker 449). Previously we showed that the electric field of the protonated histidines at low extracellular pH (pHe) creates a potential barrier for K+ ions just outside the pore that hinders their exit from the binding site controlling inactivation (control site) thereby slowing inactivation kinetics. Here we examined the effects of extracellular potassium [K+]e and pHe on the rate of inactivation of Kv1.3 using whole-cell patch-clamp. We found that in 150 mM [K+]e inactivation was accelerated upon switching to pHe 5.5 as opposed to the slowing at 5 mM [K+]e. The transition from slowing to acceleration occurred at 40 mM [K+]e, whereas this "turning point" was at 20 mM [K+]e for inward currents. The rate of entry of Ba(2+) ions from the extracellular space to the control site was significantly slowed by low pHe in wild-type hKv1.3, but it was insensitive to pH(e) in H399K and H399L mutants. Based on these observations we expanded our model and propose that the potential barrier created by the protonated histidines impedes the passage of K+ ions between the extracellular medium and the control site in both directions and the effect on inactivation rate (acceleration or slowing) depends on the relative contribution of filling from the extracellular and intracellular sides.     

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.