Membrane stretch slows the concerted step prior to opening in a Kv channel

In the simplest model of channel mechanosensitivity, expanded states are favored by stretch. We showed previously that stretch accelerates voltage-dependent activation and slow inactivation in a Kv channel, but whether these transitions involve expansions is unknown. Thus, while voltage-gated channels are mechanosensitive, it is not clear whether the simplest model applies. For Kv pore opening steps, however, there is excellent evidence for concerted expansion motions. To ask how these motions respond to stretch, therefore, we have used a Kv1 mutant, Shaker ILT, in which the step immediately prior to opening is rate limiting for voltage-dependent current. Macroscopic currents were measured in oocyte patches before, during, and after stretch. Invariably, and directly counter to prediction for expansion-derived free energy, ILT current activation (which is limited by the concerted step prior to pore opening) slowed with stretch and the g(V) curve reversibly right shifted. In WTIR (wild type, inactivation removed), the g(V) (which reflects independent voltage sensor motions) is left shifted. Stretch-induced slowing of ILT activation was fully accounted for by a decreased basic forward rate, with no change of gating charge. We suggest that for the highly cooperative motions of ILT activation, stretch-induced disordering of the lipid channel interface may yield an entropy increase that dominates over any stretch facilitation of expanded states. Since tail current tau(V) reports on the opposite (closing) motions, ILT and WTIR tau(V)(tail) were determined, but the stretch responses were too complex to shed much light. Shaw is the Kv3 whose voltage sensor, introduced into Shaker, forms the chimera that ILT mimics. Since Shaw2 F335A activation was reportedly a first-order concerted transition, we thought its activation might, like ILT's, slow with stretch. However, Shaw2 F335A activation proved to be sigmoid shaped, so its rate-limiting transition was not a concerted pore-opening transition. Moreover, stretch, via an unidentified non-rate-limiting transition, augmented steady-state current in Shaw2 F335A. Since putative area expansion and compaction during ILT pore opening and closing were not the energetically consequential determinants of stretch modulation, models incorporating fine details of bilayer structural forces will probably be needed to explain how, for Kv channels, bilayer stretch slows some transitions while accelerating others.     

Mechanosensitivity of N-type calcium channel currents

Mechanosensitivity in voltage-gated calcium channels could be an asset to calcium signaling in healthy cells or a liability during trauma. Recombinant N-type channels expressed in HEK cells revealed a spectrum of mechano-responses. When hydrostatic pressure inflated cells under whole-cell clamp, capacitance was unchanged, but peak current reversibly increased ~1.5-fold, correlating with inflation, not applied pressure. Additionally, stretch transiently increased the open-state inactivation rate, irreversibly increased the closed-state inactivation rate, and left-shifted inactivation without affecting the activation curve or rate. Irreversible mechano-responses proved to be mechanically accelerated components of run-down; they were not evident in cell-attached recordings where, however, reversible stretch-induced increases in peak current persisted. T-type channels (alpha(1I) subunit only) were mechano-insensitive when expressed alone or when coexpressed with N-type channels (alpha(1B) and two auxiliary subunits) and costimulated with stretch that augmented N-type current. Along with the cell-attached results, this differential effect indicates that N-type mechanosensitivity did not depend on the recording situation. The insensitivity of T-type currents to stretch suggested that N-type mechano-responses might arise from primary/auxiliary subunit interactions. However, in single-channel recordings, N-type currents exhibited reversible stretch-induced increases in NP(o) whether the alpha(1B) subunit was expressed alone or with auxiliary subunits. These findings set the stage for the molecular dissection of calcium current mechanosensitivity.     

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.     

A high-Na(+) conduction state during recovery from inactivation in the K(+) channel Kv1.5

Na(+) conductance through cloned K(+) channels has previously allowed characterization of inactivation and K(+) binding within the pore, and here we have used Na(+) permeation to study recovery from C-type inactivation in human Kv1.5 channels. Replacing K(+) in the solutions with Na(+) allows complete Kv1.5 inactivation and alters the recovery. The inactivated state is nonconducting for K(+) but has a Na(+) conductance of 13% of the open state. During recovery, inactivated channels progress to a higher Na(+) conductance state (R) in a voltage-dependent manner before deactivating to closed-inactivated states. Channels finally recover from inactivation in the closed configuration. In the R state channels can be reactivated and exhibit supernormal Na(+) currents with a slow biexponential inactivation. Results suggest two pathways for entry to the inactivated state and a pore conformation, perhaps with a higher Na(+) affinity than the open state. The rate of recovery from inactivation is modulated by Na(+)(o) such that 135 mM Na(+)(o) promotes the recovery to normal closed, rather than closed-inactivated states. A kinetic model of recovery that assumes a highly Na(+)-permeable state and deactivation to closed-inactivated and normal closed states at negative voltages can account for the results. Thus these data offer insight into how Kv1. 5 channels recover their resting conformation after inactivation and how ionic conditions can modify recovery rates and pathways.     

Involvement of Kv1.1 and Nav1.5 in proliferation of gastric epithelial cells

In the present study, patch clamp experiments demonstrated the expression of multiple ionic currents, including a Ba2+-sensitive inward rectifier K+ current (IKir), a 4-aminopyridine- (4-AP) sensitive delayed rectifier K+ current (IKDR), and a nifedipine-sensitive, tetrodotoxin-resistant inward Na+ current (INa.TTXR) in the non-transformed rat gastric epithelial cell line RGM-1. RT-PCR revealed molecular identities of mRNAs for the functional ionic currents, including Kir1.2 for IKir, Kv1.1, Kv1.6, and Kv2.1 for IKDR, and Nav1.5 for INa.TTXR. Pharmacologic blockade of Kv and Nav, but not Kir, suppressed RGM-1 cell proliferation. To further elucidate which subtypes of the ion channels were involved in cell proliferation, RNA interference was employed to knockdown specific gene expression. Downregulation of Kv1.1 or Nav1.5 by RNA interference suppressed RGM-1 cell proliferation. To conclude, our study is the first to delineate the expression of ion channels and their functions as growth modulators in gastric epithelial cells.     

Inhibition of voltage-gated K+ channels in dendritic cells by rapamycin

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.     

Gating charge immobilization in Kv4.2 channels: the basis of closed-state inactivation

Kv4 channels mediate the somatodendritic A-type K+ current (I(SA)) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin (CTX)-sensitive Kv4.2 channel, which enabled us to obtain the first measurements of Kv4.2 gating currents after blocking K+ conduction with CTX (Dougherty and Covarrubias. 2006J. Gen. Physiol. 128:745-753). Here, we exploited this approach further to investigate the mechanism that links closed-state inactivation to slow Q-immobilization in Kv4 channels. The main observations revealed profound Q-immobilization at steady-state over a range of hyperpolarized voltages (-110 to -75 mV). Depolarization in this range moves <5% of the observable Q associated with activation and is insufficient to open the channels significantly. The kinetics and voltage dependence of Q-immobilization and ionic current inactivation between -153 and -47 mV are similar and independent of the channel's proximal N-terminal region (residues 2-40). A coupled state diagram of closed-state inactivation with a quasi-absorbing inactivated state explained the results from ionic and gating current experiments globally. We conclude that Q-immobilization and closed-state inactivation at hyperpolarized voltages are two manifestations of the same process in Kv4.2 channels, and propose that inactivation in the absence of N- and P/C-type mechanisms involves desensitization to voltage resulting from a slow conformational change of the voltage sensors, which renders the channel's main activation gate reluctant to open.     

Comparison of the endogenous IK currents in rat hippocampal neurons and cloned Kv2.1 channels in CHO cells

The Kv2.1 potassium channel is a principal component of the delayed rectifier I(K) current in the pyramidal neurons of cortex and hippocampus. We used whole-cell patch-clamp recording techniques to systemically compare the electrophysiological properties between the native neuronal I(K) current of cultured rat hippocampal neurons and the cloned Kv2.1 channel currents in the CHO cells. The slope factors for the activation curves of both currents obtained at different prepulse holding potentials and holding times were similar, suggesting similar voltage-dependent gating. However, the half-maximal activation voltage for I(K) was approximately 20 mV more negative than the Kv2.1 channel in CHO cells at a given prepulse condition, indicating that the neuronal I(K) current had a lower threshold for activation than that of the Kv2.1 channel. In addition, the neuronal I(K) showed a stronger holding membrane potential and holding time-dependence than Kv2.1. The Kv2.1 channel gave a U-shaped inactivation, while the I(K) current did not. The I(K) current also had much stronger voltage-dependent inactivation than Kv2.1. These results imply that the neuronal factors could make Kv2.1 channels easier to activate. The information obtained from these comparative studies help elucidate the mechanism of molecular regulation of the native neuronal I(K) current in neurons.     

Modifications of human cardiac sodium channel gating by UVA light

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.     

Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor

The action of tyrphostin AG-1478, a potentprotein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels(Kv1.5) stably expressed in Chinese hamster ovary cells wasinvestigated using the whole cell patch-clamp technique. AG-1478rapidly and reversibly inhibited Kv1.5 currents at 50 mV in aconcentration-dependent manner with an IC₅₀ of 9.82 µM.AG-1478 accelerated the decay rate of inactivation of Kv1.5 currentswithout modifying the kinetics of current activation. Pretreatment withthe structurally dissimilar PTK inhibitors (genistein and lavendustinA) had no effect on the AG-1478-induced inhibition of Kv1.5 and did notmodify the AG-1478-induced current kinetics. The rate constants forbinding and unbinding of AG-1478 were 1.46 µM¹ · s¹ and 10.19 s¹, respectively. The AG-1478-induced inhibition of Kv1.5channels was voltage dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG-1478 produced no significant effect on the steady-state activation or inactivation curves. AG-1478 slowed the deactivation timecourse, resulting in a tail crossover phenomenon. Inhibition of Kv1.5by AG-1478 was use dependent. The present results suggest that AG-1478acts directly on Kv1.5 currents as an open-channel blocker andindependently of the effects of AG-1478 on PTK activity.

     

Rapid induction of P/C-type inactivation is the mechanism for acid-induced K+ current inhibition

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H(+)(o)-induced current inhibition by taking advantage of Na(+) permeation through inactivated channels. In hKv1.5, H(+)(o) inhibited open-state Na(+) current with a similar potency to K(+) current, but had little effect on the amplitude of inactivated-state Na(+) current. In support of inactivation as the mechanism for the current reduction, Na(+) current through noninactivating hKv1.5-R487V channels was not affected by [H(+)(o)]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H(+)(o). These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na(+) currents but the maintained presence of slow Na(+) tail currents, combined with changes in the Na(+) tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na(+) current at low pH.     

Coupled left-shift of Nav channels: modeling the Na+-loading and dysfunctional excitability of damaged axons

Injury to neural tissue renders voltage-gated Na(+) (Nav) channels leaky. Even mild axonal trauma initiates Na(+) -loading, leading to secondary Ca(2+)-loading and white matter degeneration. The nodal isoform is Nav1.6 and for Nav1.6-expressing HEK-cells, traumatic whole cell stretch causes an immediate tetrodotoxin-sensitive Na(+)-leak. In stretch-damaged oocyte patches, Nav1.6 current undergoes damage-intensity dependent hyperpolarizing- (left-) shifts, but whether left-shift underlies injured-axon Nav-leak is uncertain. Nav1.6 inactivation (availability) is kinetically limited by (coupled to) Nav activation, yielding coupled left-shift (CLS) of the two processes: CLS should move the steady-state Nav1.6 "window conductance" closer to typical firing thresholds. Here we simulated excitability and ion homeostasis in free-running nodes of Ranvier to assess if hallmark injured-axon behaviors-Na(+)-loading, ectopic excitation, propagation block-would occur with Nav-CLS. Intact/traumatized axolemma ratios were varied, and for some simulations Na/K pumps were included, with varied in/outside volumes. We simulated saltatory propagation with one mid-axon node variously traumatized. While dissipating the [Na(+)] gradient and hyperactivating the Na/K pump, Nav-CLS generated neuropathic pain-like ectopic bursts. Depending on CLS magnitude, fraction of Nav channels affected, and pump intensity, tonic or burst firing or nodal inexcitability occurred, with [Na(+)] and [K(+)] fluctuating. Severe CLS-induced inexcitability did not preclude Na(+)-loading; in fact, the steady-state Na(+)-leaks elicited large pump currents. At a mid-axon node, mild CLS perturbed normal anterograde propagation, and severe CLS blocked saltatory propagation. These results suggest that in damaged excitable cells, Nav-CLS could initiate cellular deterioration with attendant hyper- or hypo-excitability. Healthy-cell versions of Nav-CLS, however, could contribute to physiological rhythmic firing.     

Gating charge immobilization caused by the transition between inactivated states in the Kv1.5 channel

Sustained Na(+) or Li(+) conductance is a feature of the inactivated state in wild-type (WT) and nonconducting Shaker and Kv1.5 channels, and has been used here to investigate the cause of off-gating charge immobilization in WT and Kv1.5-W472F nonconducting mutant channels. Off-gating immobilization in response to brief pulses in cells perfused with NMG/NMG is the result of a more negative voltage dependence of charge recovery (V(1/2) is -96 mV) compared with on-gating charge movement (V(1/2) is -6.3 mV). This shift is known to be associated with slow inactivation in Shaker channels and the disparity is reduced by 40 mV, or approximately 50% in the presence of 135 mM Cs. Off-gating charge immobilization is voltage-dependent with a V(1/2) of -12 mV, and correlates well with the development of Na(+) conductance on repolarization through C-type inactivated channels (V(1/2) is -11 mV). As well, the time-dependent development of the inward Na(+) tail current and gating charge immobilization after depolarizing pulses of different durations has the same time constant (tau = 2.7 ms). These results indicate that in Kv1.5 channels the transition to a stable C-type inactivated state takes only 2-3 ms and results in strong charge immobilization in the absence of Group IA metal cations, or even in the presence of Na. Inclusion of low concentrations of Cs delays the appearance of Na(+) tail currents in WT channels, prevents transition to inactivated states in Kv1.5-W472F nonconducting mutant channels, and removes charge immobilization. Higher concentrations of Cs are able to modulate the deactivating transition in Kv1.5 channels and prevent the residual slowing of charge return.     

Dual stretch responses of mHCN2 pacemaker channels: accelerated activation, accelerated deactivation

Mechanoelectric feedback in heart and smooth muscle is thought to depend on diverse channels that afford myocytes a mechanosensitive cation conductance. Voltage-gated channels (e.g., Kv1) are stretch sensitive, but the only voltage-gated channels that are cation permeant, the pacemaker or HCN (hyperpolarization-activated cyclic nucleotide-gated) channels, have not been tested. To assess if HCN channels could contribute to a mechanosensitive cation conductance, we recorded I(HCN) in cell-attached oocyte patches before, during, and after stretch for a range of voltage protocols. I(mHCN2) has voltage-dependent and instantaneous components; only the former was stretch sensitive. Stretch reversibly accelerated hyperpolarization-induced I(mHCN2) activation (likewise for I(spHCN)) and depolarization-induced deactivation. HCN channels (like Kv1 channels) undergo mode-switch transitions that render their activation midpoints voltage history dependent. The result, as seen from sawtooth clamp, is a pronounced hysteresis. During sawtooth clamp, stretch increased current magnitudes and altered the hysteresis pattern consistent with stretch-accelerated activation and deactivation. I(mHCN2) responses to step protocols indicated that at least two transitions were mechanosensitive: an unspecified rate-limiting transition along the hyperpolarization-driven path, mode I(closed)-->mode II(open), and depolarization-induced deactivation (from mode I(open) and/or from mode II(open)). How might this affect cardiac rhythmicity? Since hysteresis patterns and "on" and "off" I(HCN) responses all changed with stretch, predictions are difficult. For an empirical overview, we therefore clamped patches to cyclic action potential waveforms. During the diastolic potential of sinoatrial node cell and Purkinje fiber waveforms, net stretch effects were frequency dependent. Stretch-inhibited (SI) I(mHCN2) dominated at low frequencies and stretch-augmented (SA) I(mHCN2) was progressively more important as frequency increased. HCN channels might therefore contribute to either SI or SA cation conductances that in turn contribute to stretch arrhythmias and other mechanoelectric feedback phenomena.     

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.     

A new mode of regulation of N-type inactivation in a Caenorhabditis elegans voltage-gated potassium channel

N-type inactivation in voltage-gated K+ (Kv) channels is a widespread means to modulate neuronal excitability and signaling. Here we have shown a novel mechanism of N-type inactivation in a Caenorhabditis elegans Kv channel. The N-terminal sequence of KVS-1 contains a domain of 22 amino acids that resembles the inactivation ball in A-type channels, which is preceded by a domain of eighteen amino acids. Wild type KVS-1 currents can be described as A-type; however, their kinetics are significantly (approximately 5-fold) slower. When the putative inactivation ball is deleted, the current becomes non-inactivating. Inactivation is restored in non-inactivating channels by diffusion of the missing inactivation domain in the cytoplasm. Deletion of the domain in front of the ball speeds inactivation kinetics approximately 5-fold. We conclude that KVS-1 is the first example of a novel type of Kv channel simultaneously possessing an N-inactivating ball preceded by an N inactivation regulatory domain (NIRD) that acts to slow down inactivation through steric mechanisms.     

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.     

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.