Presence of the Kv1.5 K(+) channel in the sinoatrial node.

The aim of this study was to establish, using immunolabeling, whether the Kv1.5 K(+) channel is present in the pacemaker of the heart, the sinoatrial (SA) node. In the atrial muscle surrounding the SA node and in the SA node itself (from guinea pig and ferret), Western blotting analysis showed a major band of the expected molecular weight, approximately 64 kD. Confocal microscopy and immunofluorescence labeling showed Kv1.5 labeling clustered in atrial muscle but punctate in the SA node. In atrial muscle, Kv1.5 labeling was closely associated with labeling of Cx43 (gap junction protein) and DPI/II (desmosomal protein), whereas in SA node Kv1.5 labeling was closely associated with labeling of DPI/II but not labeling of Cx43 (absent in the SA node) or Cx45 (another gap junction protein present in the SA node). Electron microscopy and immunogold labeling showed that the Kv1.5 labeling in atrial muscle is preferentially associated with desmosomes rather than gap junctions.     

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.     

Two pore residues mediate acidosis-induced enhancement of C-type inactivation of the Kv1.4 K(+) channel

Acidosis inhibits current through the Kv1.4 K(+) channel, perhaps as a result of enhancement of C-type inactivation. The mechanism of action of acidosis on C-type inactivation has been studied. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Delta2-146) was expressed in Xenopus oocytes, and currents were recorded using two-microelectrode voltage clamp. Acidosis increased fKv1.4 Delta2-146 C-type inactivation. Replacement of a pore histidine with cysteine (H508C) abolished the increase. Application of positively charged thiol-specific methanethiosulfonate to fKv1.4 Delta2-146 H508C increased C-type inactivation, mimicking the effect of acidosis. Replacement of a pore lysine with cysteine (K532C) abolished the acidosis-induced increase of C-type inactivation. A model of the Kv1.4 pore, based on the crystal structure of KcsA, shows that H508 and K532 lie close together. It is suggested that the acidosis-induced increase of C-type inactivation involves the charge on H508 and K532.     

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.     

A trapped intracellular cation modulates K+ channel recovery from slow inactivation

Upon depolarization, many voltage-gated potassium channels undergo a time-dependent decrease in conductance known as inactivation. Both entry of channels into an inactivated state and recovery from this state govern cellular excitability. In this study, we show that recovery from slow inactivation is regulated by intracellular permeant cations. When inactivated channels are hyperpolarized, closure of the activation gate traps a cation between the activation and inactivation gates. The identity of the trapped cation determines the rate of recovery, and the ability of cations to promote recovery follows the rank order K+ > NH4+ > Rb+ > Cs+ > Na+, TMA. The striking similarity between this rank order and that for single channel conductance suggests that these two processes share a common feature. We propose that the rate of recovery from slow inactivation is determined by the ability of entrapped cations to move into a binding site in the channel's selectivity filter, and refilling of this site is required for recovery.     

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.     

Changes in the inactivation of rat Kv1.4 K(+) channels induced by varying the number of inactivation particles

N-type inactivation of rat Kv1.4 channels with one, two, or four inactivation balls was investigated using homogeneous populations of channels expressed in Xenopus oocytes. Tandem dimeric and tetrameric constructs of Kv1.4 were made. Channels encoded by tandem cDNAs Kv1. 4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) have two or only one tethered inactivation ball, respectively, whereas Kv1.4 itself encodes channels having four inactivation balls. The time constants for inactivation of macroscopic currents were increased significantly as the number of inactivation balls was decreased, whereas the time constants for recovery from inactivation were not modified. The ratios of the rate constants of inactivation (k(inact)) of Kv1.4-Kv1.4Delta1-145 and Kv1.4-[Kv1.4Delta1-145](3) channels to that of the Kv1.4 channel were 0.65 and 0.4, respectively, whereas the ratios of the rate constant of recovery (k(rec)) of these channels to that of Kv1.4 were almost unity. The rate constants k(inact) for channels having two and four inactivation balls are smaller than those that would be expected if inactivation balls on each channel are independent, suggesting some interaction occurs between inactivation balls. Furthermore, noninactivating current became apparent as the number of inactivation balls on a channel was decreased.     

Altered state dependence of c-type inactivation in the long and short forms of human Kv1.5

Evidence from both human and murine cardiomyocytes suggests that truncated isoforms of Kv1.5 can be expressed in vivo. Using whole-cell patch-clamp recordings, we have characterized the activation and inactivation properties of Kv1.5DeltaN209, a naturally occurring short form of human Kv1.5 that lacks roughly 75% of the T1 domain. When expressed in HEK 293 cells, this truncated channel exhibited a V(1/2) of -19.5 +/- 0.9 mV for activation and -35.7 +/- 0.7 mV for inactivation, compared with a V(1/2) of -11.2 +/- 0.3 mV for activation and -0.9 +/- 1.6 mV for inactivation in full-length Kv.15. Kv1.5DeltaN209 channels exhibited several features rarely observed in voltage-gated K(+) channels and absent in full-length Kv1.5, including a U-shaped voltage dependence of inactivation and "excessive cumulative inactivation," in which a train of repetitive depolarizations resulted in greater inactivation than a continuous pulse. Kv1.5DeltaN209 also exhibited a stronger voltage dependence to recovery from inactivation, with the time to half-recovery changing e-fold over 30 mV compared with 66 mV in full-length Kv1.5. During trains of human action potential voltage clamps, Kv1.5DeltaN209 showed 30-35% greater accumulated inactivation than full-length Kv1.5. These results can be explained with a model based on an allosteric model of inactivation in Kv2.1 (Klemic, K.G., C.-C. Shieh, G.E. Kirsch, and S.W. Jones. 1998. Biophys. J. 74:1779-1789) in which an absence of the NH(2) terminus results in accelerated inactivation from closed states relative to full-length Kv1.5. We suggest that differential expression of isoforms of Kv1.5 may contribute to K(+) current diversity in human heart and many other tissues.     

Cell-cell contact between adult rat cardiac myocytes regulates Kv1.5 and Kv4.2 K+ channel mRNA expression

Regulation of voltage-gatedK⁺ channel genes represents animportant mechanism for modulating cardiac excitability. Here we demonstrate that expression of twoK⁺ channel mRNAs is reciprocallycontrolled by cell-cell interactions between adult cardiac myocytes. Itis shown that culturing acutely dissociated rat ventricular myocytesfor 3 h results in a dramatic downregulation of Kv1.5 mRNA and a modestupregulation of Kv4.2 mRNA. These effects are specific, because similarchanges are not detected with other channel mRNAs. Increasing myocytedensity promotes maintenance of Kv1.5 gene expression, whereas Kv4.2mRNA expression was found to be inversely proportional to cell density. Conditioned culture medium did not mimic the effects of high cell density. However, paraformaldehyde-fixed myocytes were comparable tolive cells in their ability to influenceK⁺ channel message levels. Thusthe reciprocal effects of cell density on the expression of Kv1.5 andKv4.2 genes are mediated by direct contact between adult cardiacmyocytes. These findings reveal for the first time that cardiac myocytegene expression is influenced by signaling induced by cell-cell contact.

     

Contributions of Kv1.2, Kv1.5 and Kv2.1 subunits to the native delayed rectifier K(+) current in rat mesenteric artery smooth muscle cells

A large array of voltage-gated K(+) channel (Kv) genes has been identified in vascular smooth muscle tissues. This molecular diversity underlies the vast repertoire of native Kv channels that regulate the excitability of vascular smooth muscle tissues. The contributions of different Kv subunit gene products to the native Kv currents are poorly understood in vascular smooth muscle cells (SMCs). In the present study, Kv subunit-specific antibodies were applied intracellularly to selectively block various Kv channel subunits and the whole-cell outward Kv currents were recorded using the patch-clamp technique in rat mesenteric artery SMCs. Anti-Kv1.2 antibody (8 microg/ml) inhibited the Kv currents by 29.2 +/- 5.9% (n = 6, P < 0.05), and anti-Kv1.5 antibody (6 microg/ml) by 24.5 +/- 2.6% (n = 7, P < 0.05). Anti-Kv2.1 antibody inhibited the Kv currents in a concentration-dependent fashion (4-20 microg/ml). Co-application of antibodies against Kv1.2 and Kv2.1 (8 microg/ml each) induced an additive inhibition of Kv currents by 42.3 +/- 3.1% (n = 7, P < 0.05). In contrast, anti-Kv1.3 antibody (6 microg/ml) did not have any effect on the native Kv current (n = 6, P > 0.05). A control antibody (anti-GIRK1) also had no effect on the native Kv currents. This study demonstrates that Kv1.2, Kv1.5, and Kv2.1 subunit genes all contribute to the formation of the native Kv channels in rat mesenteric artery SMCs.     

Closing in on the resting state of the Shaker K(+) channel

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.     

Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.     

State-dependent inactivation of the Kv3 potassium channel.

Inactivation of Kv3 (Kv1.3) delayed rectifier potassium channels was studied in the Xenopus oocyte expression system. These channels inactivate slowly during a long depolarizing pulse. In addition, inactivation accumulates in response to a series of short depolarizing pulses (cumulative inactivation), although no significant inactivation occurs within each short pulse. The extent of cumulative inactivation does not depend on the voltage during the depolarizing pulse, but it does vary in a biphasic manner as a function of the interpulse duration. Furthermore, the rate of cumulative inactivation is influenced by changing the rate of deactivation. These data are consistent with a model in which Kv3 channel inactivation is a state-dependent and voltage-independent process. Macroscopic and single channel experiments indicate that inactivation can occur from a closed (silent) state before channel opening. That is, channels need not open to inactivate. The transition that leads to the inactivated state from the silent state is, in fact, severalfold faster then the observed inactivation of current during long depolarizing pulses. Long pulse-induced inactivation appears to be slow, because its rate is limited by the probability that channels are in the open state, rather than in the silent state from which they can inactivate. External potassium and external calcium ions alter the rates of cumulative and long pulse-induced inactivation, suggesting that antagonistic potassium and calcium binding steps are involved in the normal gating of the channel.     

Discovery of triarylethanolamine inhibitors of the Kv1.5 potassium channel

A series of triarylethanolamine inhibitors of the Kv1.5 potassium channel have been prepared and evaluated for their effects in vitro and in vivo. The structure–activity relationship (SAR) studies described herein led to the development of potent, selective and orally active inhibitors of Kv1.5.     

Aryl sulfonamido tetralin inhibitors of the Kv1.5 ion channel

Aryl sulfonamido tetralins based on lead compound 2a were synthesized and evaluated for Kv1.5 inhibitory activity. Several compounds having IC₅₀ values less then 0.1 μM were identified. Kv1.5 inhibitors have the potential to be atrium-selective agents for the treatment of atrial fibrillation.     

Aryl sulfonamido indane inhibitors of the Kv1.5 ion channel

A collection of aryl sulfonamido indanes based on the lead compound 1 was synthesized and evaluated for Kv1.5 inhibitory activity. Kv1.5 inhibitors have the potential to be atrium-selective agents for treatment of atrial fibrillation. (1R,2R)-1 has an IC(50) of 0.033microM against Kv1.5 and is selective against other cardiac ion channels, including hERG.     

A mutant KcsA K(+) channel with altered conduction properties and selectivity filter ion distribution

The selectivity filter of K(+) channels is comprised of a linear queue of four equal-spaced ion-binding sites spanning a distance of 12A. Each site is formed of eight oxygen atoms from the protein. The first three sites, numbered 1-3 from the extracellular side, are made of exclusively main-chain carbonyl oxygen atoms. The fourth site, closest to the intracellular side, is made of four main-chain carbonyl oxygen atoms and four threonine side-chain hydroxyl oxygen atoms. Here we characterize the effects of mutating the threonine to cysteine on the distribution of ions in the selectivity filter and on the conduction of ions through the filter. The mutation influences the occupancy of K(+) at sites 2 and 4 and it reduces the maximum rate of conduction in the limit of high K(+) concentration. The mutation does not affect the conduction of Rb(+). These results can be understood in the context of a conduction mechanism in which a pair of K ions switch between energetically balanced 1,3 and 2,4 configurations.