Upregulation of Kv 1.4 protein and gene expression after chronic spinal cord injury.

After spinal cord injury (SCI), white matter tracts are characterized by demyelination and increased sensitivity to the K(+) channel blocker 4-aminopyridine (4-AP). These effects appear to contribute to neurological impairment after SCI, although the molecular changes in K(+) channel subunit expression remain poorly understood. We examined changes in gene expression of the 4-AP-sensitive voltage-gated K(+) channel Kv 1.4 after chronic SCI in the rat. Quantitative immunoblotting showed that Kv 1.4 protein was significantly increased at 6 weeks, but not at 1 week, after SCI in spinal cord white matter. Kv 1.4 was localized to astrocytes, oligodendrocytes, and oligodendrocyte progenitor cells but not to axons in both the normal and the injured spinal cord white matter. Because glial cells proliferate after SCI, we used immunogold electron microscopy to quantify Kv 1.4 protein in individual glial cells and found a sixfold increase of Kv 1.4 in cells of the oligodendrocyte lineage after chronic injury. Finally, quantitative in situ hybridization showed that Kv 1.4 mRNA was significantly upregulated in spinal cord white matter, but not gray matter, after SCI. In summary, we show that Kv 1.4 is expressed in glial cells and not in axons in the rat spinal cord white matter and that its expression is markedly increased in cells of the oligodendrocyte lineage after chronic SCI. Given that K(+) channels play a role in glial cell proliferation, cells exhibiting changes in Kv 1.4 expression may represent proliferating oligodendroglia in the chronically injured spinal cord.     

Delayed rectifier currents in rat globus pallidus neurons are attributable to Kv2.1 and Kv3.1/3.2 K(+) channels.

The symptoms of Parkinson disease are thought to result in part from increased burst activity in globus pallidus neurons. To gain a better understanding of the factors governing this activity, we studied delayed rectifier K(+) conductances in acutely isolated rat globus pallidus (GP) neurons, using whole-cell voltage-clamp and single-cell RT-PCR techniques. From a holding potential of -40 mV, depolarizing voltage steps in identified GP neurons evoked slowly inactivating K(+) currents. Analysis of the tail currents revealed rapidly and slowly deactivating currents of similar amplitude. The fast component of the current deactivated with a time constant of 11. 1 +/- 0.8 msec at -40 mV and was blocked by micromolar concentrations of 4-AP and TEA (K(D) approximately 140 microM). The slow component of the current deactivated with a time constant of 89 +/- 10 microseconds at -40 mV and was less sensitive to TEA (K(D) = 0.8 mM) and 4-AP (K(D) approximately 6 mM). Organic antagonists of Kv1 family channels had little or no effect on somatic currents. These properties are consistent with the hypothesis that the rapidly deactivating current is attributable to Kv3.1/3.2 channels and the slowly deactivating current to Kv2.1-containing channels. Semiquantitative single-cell RT-PCR analysis of Kv3 and Kv2 family mRNAs supported this conclusion. An alteration in the balance of these two channel types could underlie the emergence of burst firing after dopamine-depleting lesions.     

Mechanism of 4-aminopyridine block of the transient outward K-current in identified Helix neuron

The block of the transient outward K-current, I(K(A)) by 4-aminopyridine (4-AP) and blood-depressing substances (BDS) was investigated in identified Helix pomatia neurons (LPa3) using the two microelectrode voltage-clamp technique. The present study shows that 4-AP inhibits I(K(A)) in snail neurons in a voltage- and concentration-dependent manner. The 4-AP block of I(K(A)) involves the block of both open and closed states of the channel, however binding to open channels is preferred. It is suggested that 4-AP have two binding sites on the identified Helix neuron. One site causes an open channel block, which affects the N-type inactivation, and binding to the second site induces closed channel block, which affects C-type inactivation. In control solution the inactivating phase of the current is biexponential, suggesting simultaneous presence of two types of inactivation. The counterplay of these mechanisms results in the crossover of the current traces recorded from control and 4-AP blocked channels. It is assumed that use-dependence does not occur through blocker 'trapping', but rather by a different mechanism. BDS had no effect on Helix I(K(A)), suggesting that transient potassium channels in LPa3 neuron are not Kv3.4 type channels.     

Characteristics of IA currents in adult rabbit cerebellar Purkinje cells

Classical conditioning the rabbit nictitating membrane involves changes in synaptic and intrinsic membrane properties of cerebellar Purkinje cell dendrites, and a 4-aminopyridine (4-AP)-sensitive potassium channel underlies these membrane properties. We characterized I(A) currents in adult, rabbit Purkinje cells to determine whether I(A) is the target channel involved in learning. Whole-cell recordings of Purkinje cell somas and dendrites revealed a fast activating and inactivating current with half maximal activation at -27.08 +/- 3.48 mV and -25.51 +/- 1.15 mV in somas and dendrites, respectively; half maximal inactivation at -58.91 +/- 2.34 mV and -49.90 +/- 2.58 mV; and a recovery time constant of 22.81 +/- 1.92 ms and 16.60 +/- 4.26 ms. Outside-out patch recordings from cerebellar Purkinje cell somas confirmed these 4-AP-sensitive currents with half maximal activation at -13.85 +/- 1.17 mV and half maximal inactivation at -55.07 +/- 5.54 mV. More importantly, there was an overlap of activation and incomplete inactivation at potentials from -60 to -40 mV, suggesting a "window" current that was responsible for subthreshold variations of membrane potential and might underlie conditioning-specific increases in Purkinje cell excitability. The potassium current was inhibited by 4-AP and by Heteropodatoxin, a specific blocker of Kv4.2 and Kv4.3 channels, but not by Stromatoxin, a blocker of Kv4.2 channels. Mouse monoclonal antibody labeling identified both Kv4.3 and Kv4.2 subunits in the granule cell layer but only Kv4.3 subunits in the molecular layer. This is the first demonstration of A-type currents in adult, rabbit Purkinje cells that may play a role in regulating membrane potential and firing frequency and comprise the target channel mediating conditioning-specific changes of excitability in rabbit Purkinje cell dendrites.     

Differential effects of K(+) channel blockers on frequency-dependent action potential broadening in supraoptic neurons

Recordings were made from magnocellular neuroendocrine cells dissociated from the supraoptic nucleus of the adult guinea pig to determine the role of voltage gated K(+) channels in controlling the duration of action potentials and in mediating frequency-dependent action potential broadening exhibited by these neurons. The K(+) channel blockers charybdotoxin (ChTx), tetraethylammonium (TEA), and 4-aminopyridine (4-AP) increased the duration of individual action potentials indicating that multiple types of K(+) channel are important in controlling action potential duration. The effect of these K(+) channel blockers was almost completely reversed by simultaneous blockade of voltage gated Ca(2+) channels with Cd(2+). Frequency-dependent action potential broadening was exhibited by these neurons during trains of action potentials elicited by membrane depolarizing current pulses presented at 10 Hz but not at 1 Hz. 4-AP but not ChTx or TEA inhibited frequency-dependent action potential broadening indicating that frequency-dependent action potential broadening is dependent on increasing steady-state inactivation of A-type K(+) channels (which are blocked by 4-AP). A model of differential contributions of voltage gated K(+) channels and voltage gated Ca(2+) channels to frequency-dependent action potential broadening, in which an increase of Ca(2+) current during each successive action potential is permitted as a result of the increasing steady-state inactivation of A-type K(+) channels, is presented.     

K+ channel alterations in the progression of experimental autoimmune encephalomyelitis

Voltage-gated K(+) (Kv) channels play critical roles not only in regulating synaptic transmission and intrinsic excitability of neurons, but also in controlling the function and proliferation of other cells in the central nervous system (CNS). The non-specific Kv channel blocker, 4-AminoPyridine (4-AP) (Dalfampridine, Ampyra?), is currently used to treat multiple sclerosis (MS), an inflammatory demyelinating disease. However, little is known how various types of Kv channels are altered in any inflammatory demyelinating diseases. By using established animal models for MS, experimental autoimmune encephalomyelitis (EAE), we report that expression and distribution patterns of Kv channels are altered in the CNS correlating with EAE severity. The juxtaparanodal (JXP) targeting of Kv1.2/Kvβ2 along myelinated axons is disrupted within demyelinated lesions in the white matter of spinal cord in EAE. Moreover, somatodendritic Kv2.1 channels in the motor neurons of lower spinal cord significantly decrease correlating with EAE severity. Interestingly, Kv1.4 expression surrounding lesions is markedly up-regulated in the initial acute phase of both EAE models. Its expression in glial fibrillary acidic protein (GFAP)-positive astrocytes further increases in the remitting phase of remitting-relapsing EAE (rrEAE), but decreases in late chronic EAE (chEAE) and the relapse of rrEAE, suggesting that Kv1.4-positive astrocytes may be neuroprotective. Taken together, our studies reveal myelin-dependent and -independent alterations of Kv channels in the progression of EAE and lay a solid foundation for future study in search of a better treatment for MS.     

Molecular physiology and modulation of somatodendritic A-type potassium channels

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.     

Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore

In Kv1.1, single point mutants found below the channel activation gate at residue V408 are associated with human episodic ataxia type-1, and impair channel function by accelerating decay of outward current during periods of membrane depolarization and channel opening. This decay is usually attributed to C-type inactivation, but here we provide evidence that this is not the case. Using voltage-clamp fluorimetry in Xenopus oocytes, and single-channel patch clamp in mouse ltk- cells, of the homologous Shaker channel (with the equivalent mutation V478A), we have determined that the mutation may cause current decay through a local effect at the activation gate, by destabilizing channel opening. We demonstrate that the effect of the mutant is similar to that of trapped 4-aminopyridine in antagonizing channel opening, as the mutation and 10 mm 4-AP had similar, nonadditive effects on fluorescence recorded from the voltage-sensitive S4 helix. We propose a model where the Kv1.1 activation gate fails to enter a stabilized open conformation, from which the channel would normally C-type inactivate. Instead, the lower pore lining helix is able to enter an activated-not-open conformation during depolarization. These results provide an understanding of the molecular etiology underlying episodic ataxia type-1 due to V408A, as well as biophysical insights into the links between the potassium channel activation gate, the voltage sensor and the selectivity filter.     

Facilitation of recovery from inactivation by external Na+ and location of the activation gate in neuronal Na+ channels.

Fast inactivation of the Na(+) channel presumably is produced by binding of the inactivating peptide (the "hinged lid") to the internal pore mouth of the activated channel. It has been shown that recovery from inactivation in Na(+) channels begins with a delay, which corresponds to deactivation of the channel, and is then followed by an exponential phase, which corresponds to unbinding of the inactivating peptide. We found that the exponential phase is approximately 1.6-fold faster in 150 mm than in 0 mm external Na(+), but the initial delays are the same. External Na(+) also increases the late steady-state Na(+) current during a step depolarization and shifts the inactivation curve accordingly but has no effect on the activation and deactivation kinetics of the current. Quantitative analysis of the data reveals that external Na(+) has the same facilitation effect on the unbinding of the bound inactivating peptide whether the channel is activated or deactivated but has no effect on the other gating processes of the channel. These findings suggest that permeating Na(+) ions directly knock off the bound inactivating peptide and that channel activation or deactivation does not affect the accessibility of the bound inactivation peptide to external Na(+). The activation gate (the key gating change transforming a Na(+)-nonconducting pore into a Na(+)-conducting one) therefore should not be located external to the inactivation gate, which presumably is already located close to the internal end of the pore.     

Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kvbeta1.1 and Kv1.4-1.1/Kvbeta1.2

Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker-like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kvbeta1.x subunits. Here we show that the human Kvbeta1.1 and Kvbeta1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kvbeta1.1 and Kv1.4-1.1/Kvbeta1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kvbeta1.1 and Kvbeta1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements.     

4-aminopyridine, a specific blocker of K(+) channels, inhibited inward Na(+) current in rat cerebellar granule cells

The effects of 4-aminopyridine (4-AP), a specific blocker of outward K(+) current, on voltage-activated transient outward K(+) current (I(K(A))) and inward Na(+) current (I(Na)) were investigated on cultured rat cerebellar granule cells using the whole cell voltage-clamp technique. At the concentration of 1-5 mM, 4-AP inhibited both I(K(A)) and I(Na). It reduced the amplitude of peak Na(+) current without significant alteration of the steady-state activation and inactivation properties. The inhibitory effect was not enhanced by repeated depolarizing pulses (0.5 or 0.1 Hz), suggesting that the binding affinity of 4-AP on Na(+) channels is state-independent. In contrast, the effect of 4-AP on Na(+) channels appeared to be voltage-dependent, the weaker inhibition occurred at more depolarization. Moreover, 4-AP slowed both the activation and inactivation kinetics of Na(+) current. These effects were similar to those induced by alpha-scorpion toxin and sea anemone toxins on Na(+) channels in other cell model. Our data demonstrate for the first time that 4-AP is able to block not only A-type K(+) channels, but also Na(+) channels in rat cerebellar granule cells. It is concluded that the inhibition exerted by 4-AP on Na(+) current likely differs from that provoked by local anesthetics. The possibility that the binding site of neurotoxin receptor 3 may be involved is discussed.     

Effects of Kv1.1 channel glycosylation on C-type inactivation and simulated action potentials

Kv1.1 channels are brain glycoproteins that play an important role in repolarization of action potentials. In previous work, we showed that lack of N-glycosylation, particularly lack of sialylation, of Kv1.1 affected its macroscopic gating properties and slowed activation and C-type inactivation kinetics and produced a depolarized shift in the steady-state activation curve. In our current study, we used single channel analysis to investigate voltage-independent C-type inactivation in both Kv1.1 and Kv1.1N207Q, a glycosylation mutant. Both channels underwent brief and long-lived closures, and the lifetime and frequency of the long-lived closed states were voltage-independent and similar for both channels. We found that, as in macroscopic measurements, Kv1.1N207Q exhibited a approximately 8 mV positive shift in its single channel fractional open time (fo) and a shallower fo-voltage slope compared with Kv1.1. Data suggested that C-type inactivation reflected the equilibration time with at least two slow voltage-independent long-lived closed states that followed the rapid activation process. In addition, data simulation indicated that the C-type inactivation process reflected the equilibration time between the open state and at least two long-lived closed states. Moreover, the faster macroscopic current decay in Kv1.1 mostly reflected a slower equilibration time in these channels as compared with Kv1.1N207Q. Finally, action potential simulations indicated that the N207Q mutation broaden the action potential and decreased the interspike interval. The shape of the action potential was not significantly affected by C-type inactivation, however, for a given channel, C-type inactivation increased the interspike interval. Data and simulations suggested that excitable cells could use differences in K(+) channel glycosylation degree as an additional mechanism to increase channel functional diversity which could modify cell excitability.     

Abnormal axonal physiology is associated with altered expression and distribution of Kv1.1 and Kv1.2 K+ channels after chronic spinal cord injury

Dysfunction of surviving axons which traverse the site of spinal cord injury (SCI) has been linked to altered sensitivity to the K+ channel blocker 4-aminopyridine (4-AP) and appears to contribute to post-traumatic neurological deficits although the underlying mechanisms remain unclear. In this study, sucrose gap electrophysiology in isolated dorsal column strips, Western blotting and confocal immunofluorescence microscopy were used to identify the K+ channels associated with axonal dysfunction after chronic (6-8 weeks postinjury) clip compresssion SCI of the thoracic cord at T7 in rats. The K+ channel blockers 4-AP (200 microM, 1 mM and 10 mM) and alpha-dendrotoxin (alpha-DTX, 500 nM) resulted in a significant relative increase in the amplitude and area of compound action potentials (CAP) recorded from chronically injured dorsal column axons in comparison with control noninjured preparations. In contrast, TEA (10 mM) and CsCl (2 mM) had similar effects on injured and control spinal cord axons. Western blotting and quantitative immunofluorescence microscopy showed increased expression of Kv1.1 and Kv1.2 K+ channel proteins on spinal cord axons following injury. In addition, Kv1.1 and Kv1.2 showed a dispersed staining pattern along injured axons in contrast to a paired juxtaparanodal localization in uninjured spinal cord axons. Furthermore, labelled alpha-DTX colocalized with Kv1.1 and Kv1.2 along axons. These findings suggest a novel mechanism of axonal dysfunction after SCI whereby an increased 4-AP- and alpha-DTX-sensitive K+ conductance, mediated in part by increased Kv1.1 and Kv1.2 K+ channel expression, contributes to abnormal axonal physiology in surviving axons.     

RNA editing of Kv1.1 channels may account for reduced ictogenic potential of 4-aminopyridine in chronic epileptic rats

In rat brain slices, the Kv channel blocker 4-aminopyridine (4-AP) induces seizure-like events. This effect is absent in slices from chronic epileptic rats generated using the kainic acid model. The reason for this phenomenon remained elusive as an altered expression level of Kv channels was ruled out as a mechanism. We recently described that the Ile400Val RNA editing of Kv1.1 generates 4-AP-insensitive Kv1 channels (Kv1.1(I400V)). We therefore hypothesized that altered RNA editing levels account for the reduced ictogenic potency of 4-AP in chronic epileptic rats. We found fourfold increased RNA editing ratios in the entorhinal cortex of chronic epileptic animals compared to healthy control animals. Electrophysiologic recordings in Xenopus oocytes revealed that the observed increased Kv1.1(I400V) editing level can in fact lead to significant loss of 4-AP sensitivity. Our data suggest that altered Kv1.1(I400V) RNA editing contributes to the reduced ictogenic potential of 4-AP in chronic epileptic rats.     

Kv3 K+ channels enable burst output in rat cerebellar Purkinje cells

The ability of cells to generate an appropriate spike output depends on a balance between membrane depolarizations and the repolarizing actions of K(+) currents. The high-voltage-activated Kv3 class of K(+) channels repolarizes Na(+) spikes to maintain high frequencies of discharge. However, little is known of the ability for these K(+) channels to shape Ca(2+) spike discharge or their ability to regulate Ca(2+) spike-dependent burst output. Here we identify the role of Kv3 K(+) channels in the regulation of Na(+) and Ca(2+) spike discharge, as well as burst output, using somatic and dendritic recordings in rat cerebellar Purkinje cells. Kv3 currents pharmacologically isolated in outside-out somatic membrane patches accounted for approximately 40% of the total K(+) current, were very fast and high voltage activating, and required more than 1 s to fully inactivate. Kv3 currents were differentiated from other tetraethylammonium-sensitive currents to establish their role in Purkinje cells under physiological conditions with current-clamp recordings. Dual somatic-dendritic recordings indicated that Kv3 channels repolarize Na(+) and Ca(2+) spikes, enabling high-frequency discharge for both types of cell output. We further show that during burst output Kv3 channels act together with large-conductance Ca(2+)-activated K(+) channels to ensure an effective coupling between Ca(2+) and Na(+) spike discharge by preventing Na(+) spike inactivation. By contributing significantly to the repolarization of Na(+) and especially Ca(2+) spikes, our data reveal a novel function for Kv3 K(+) channels in the maintenance of high-frequency burst output for cerebellar Purkinje cells.     

Effects of 4-aminopyridine on sharp wave-ripples in rat hippocampal slices

Sharp wave-ripple complexes (SPW-Rs) are characterized by approximately 60 ms field potential transients superimposed by ripple oscillations of approximately 200 Hz. In chronic epileptic rodents and humans, faster ripples have been recorded showing frequencies of up to 500 Hz. In this study, we tested whether the blockade of K currents by 4-aminopyridine (4-AP) contribute to the generation of high-frequency ripples, as changes in K channel expression have been observed in chronic epileptic tissue. We showed that 4-AP significantly increased the amplitudes and incidence of induced SPW-Rs without significantly changing their ripple frequency. alpha-Dendrotoxin or BDS-I did not mimick these changes suggesting that 4-AP acts via Kv1.4 channels. Thus, the incidence of SPW-Rs, but not the ripple frequency is regulated by 4-AP-sensitive potassium currents.