Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.     

Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons.     

Differential expression of Kv3.1b and Kv3.2 potassium channel subunits in interneurons of the basolateral amygdala

The expression of Kv3.1 and Kv3.2 voltage-gated potassium channel subunits appears to be critical for high-frequency firing of many neuronal populations. In the cortex these subunits are mainly associated with fast-firing GABAergic interneurons containing parvalbumin or somatostatin. Since the basolateral nuclear complex of the amygdala contains similar interneurons, it is of interest to determine if these potassium channel subunits are expressed in these same interneuronal subpopulations. To investigate this issue, peroxidase and dual-labeling fluorescence immunohistochemistry combined with confocal laser scanning microscopy was used to determine which interneuronal subpopulations in the basolateral nuclear complex of the rat amygdala express Kv3.1b and Kv3.2 subunits. Antibodies to parvalbumin, somatostatin, calretinin, and cholecystokinin were used to label separate subsets of basolateral amygdalar interneurons. Examination of immunoperoxidase preparations suggested that the expression of both channels was restricted to nonpyramidal interneurons in the basolateral amygdala. Somata and proximal dendrites were intensely-stained, and axon terminals arising from presumptive basket cells and chandelier cells were lightly stained. Immunofluorescence observations revealed that parvalbumin+ neurons were the main interneuronal subpopulation expressing the Kv3.1b potassium channel subunit in the basolateral amygdala. More than 92-96% of parvalbumin+ neurons were Kv3.1b+, depending on the nucleus. These parvalbumin+/Kv3.1b+ double-labeled cells constituted 90-99% of all Kv3.1b+ neurons. Parvalbumin+ neurons were also the main interneuronal subpopulation expressing the Kv3.2 potassium channel subunit. More than 67-78% of parvalbumin+ neurons were Kv3.2+, depending on the nucleus. However, these parvalbumin+/Kv3.2+ double-labeled cells constituted only 71-81% of all Kv3.2+ neurons. Most of the remaining neurons with significant levels of the Kv3.2 subunit were somatostatin+ interneurons. These Kv3.2-containing somatostatin+ interneurons constituted 27-50% of the somatostatin+ population, depending on the nucleus in question. These data suggest that both fast-firing and burst-firing parvalbumin+ interneurons in the basolateral amygdala express the Kv3.1b subunit. The significance of Kv3.2 expression in some parvalbumin+ and somatostatin+ interneurons remains to be determined.     

Acoustic environment determines phosphorylation state of the Kv3.1 potassium channel in auditory neurons

Sound localization by auditory brainstem nuclei relies on the detection of microsecond interaural differences in action potentials that encode sound volume and timing. Neurons in these nuclei express high amounts of the Kv3.1 potassium channel, which allows them to fire at high frequencies with short-duration action potentials. Using computational modeling, we show that high amounts of Kv3.1 current decrease the timing accuracy of action potentials but enable neurons to follow high-frequency stimuli. The Kv3.1b channel is regulated by protein kinase C (PKC), which decreases current amplitude. Here we show that in a quiet environment, Kv3.1b is basally phosphorylated in rat brainstem neurons but is rapidly dephosphorylated in response to high-frequency auditory or synaptic stimulation. Dephosphorylation of the channel produced an increase in Kv3.1 current, facilitating high-frequency spiking. Our results indicate that the intrinsic electrical properties of auditory neurons are rapidly modified to adjust to the ambient acoustic environment.     

Visual experience regulates Kv3.1b and Kv3.2 expression in developing rat visual cortex

Among the GABAergic neocortical interneurons, parvalbumin-containing fast-spiking (FS) basket cells are essential mediators of feed-forward inhibition, network synchrony and oscillations, and timing of the critical period for sensory plasticity. The FS phenotype matures after birth. It depends on the expression of the voltage-gated potassium channels Kv3.1b/3.2 which mediate the fast membrane repolarization necessary for firing fast action potentials at high frequencies. We have now tested in rat visual cortex if visual deprivation affects the Kv3 expression. During normal development, Kv3.1b/3.2 mRNA and protein expression increased in rat visual cortex reaching adult levels around P20. Dark rearing from birth neither prevented nor delayed the upregulation. Rather unexpectedly, the expression of Kv3.1b protein and Kv3.2 mRNA and protein increased to higher levels from the third postnatal week onwards. Triple-labeling revealed that in dark-reared visual cortex Kv3.2 was upregulated in parvalbuminergic interneurons in supragranular layers which in normal animals rarely display Kv3.2 expression. Recovery from dark rearing normalized Kv3.2 expression. This showed that visual experience influences the Kv3 expression. The results suggest that an altered expression of Kv3 channels affects the functional properties of FS neurons, and may contribute to the deficits in inhibition observed in the sensory-deprived cortex.     

Specific and rapid effects of acoustic stimulation on the tonotopic distribution of Kv3.1b potassium channels in the adult rat

Recent studies have demonstrated that total cellular levels of voltage-gated potassium channel subunits can change on a time scale of minutes in acute slices and cultured neurons, raising the possibility that rapid changes in the abundance of channel proteins contribute to experience-dependent plasticity in vivo. In order to investigate this possibility, we took advantage of the medial nucleus of the trapezoid body (MNTB) sound localization circuit, which contains neurons that precisely phase-lock their action potentials to rapid temporal fluctuations in the acoustic waveform. Previous work has demonstrated that the ability of these neurons to follow high-frequency stimuli depends critically upon whether they express adequate amounts of the potassium channel subunit Kv3.1. To test the hypothesis that net amounts of Kv3.1 protein would be rapidly upregulated when animals are exposed to sounds that require high frequency firing for accurate encoding, we briefly exposed adult rats to acoustic environments that varied according to carrier frequency and amplitude modulation (AM) rate. Using an antibody directed at the cytoplasmic C-terminus of Kv3.1b (the adult splice isoform of Kv3.1), we found that total cellular levels of Kv3.1b protein—as well as the tonotopic distribution of Kv3.1b-labeled cells—was significantly altered following 30 min of exposure to rapidly modulated (400 Hz) sounds relative to slowly modulated (0–40 Hz, 60 Hz) sounds. These results provide direct evidence that net amounts of Kv3.1b protein can change on a time scale of minutes in response to stimulus-driven synaptic activity, permitting auditory neurons to actively adapt their complement of ion channels to changes in the acoustic environment.     

Hyperalgesia in mice lacking the Kv1.1 potassium channel gene

Hyperalgesia and morphine induced antinociception were measured in mice lacking the gene for the Shaker-like voltage-gated potassium channel Kv1.1 alpha subunit. The effects of varying gene dosage were studied by comparing homozygous null (−/−) versus heterozygous (±) and wildtype (+/+) littermates. Hyperalgesia was measured using the paw flick assay, hot plate assay and formalin induced hind paw licking. It was observed that null mutant animals had significantly shorter latencies to response in the paw flick (36%) and hot plate (27%) assays while their licking times after hind paw injection of formalin was increased in both the first (74%) and second (65%) phases of the response compared to wildtype controls. Morphine induced antinociception in Kv1.1 null mutant animals was blunted. These studies indicate that Kv1.1 plays an important role in nociceptive and antinociceptive signaling pathways.     

Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1

Sympathetic preganglionic neurones located in the intermediolateral cell column (IML) are subject to inputs descending from higher brain regions, as well as strong influences from local interneurones. Since interneurones in the IML have been rarely studied directly we examined their electrophysiological and anatomical properties. Whole cell patch clamp recordings were made from neurones in the IML of 250 microM slices of the thoracic spinal cord of the rat at room temperature. Action potential durations of interneurones (4.2+/-0.1 ms) were strikingly shorter than those of sympathetic preganglionic neurones (9.4+/-0.2 ms) due to a more rapid repolarisation phase. Low concentrations of tetraethylammonium chloride (TEA) (0.5 mM) or 4-aminopyridine (4-AP) (30 microM) affected interneurones but not sympathetic preganglionic neurones by prolonging the action potential repolarisation as well as decreasing both the afterhypolarisation amplitude and firing frequency. Following recordings, neurones sensitive to TEA and 4-AP were confirmed histologically as interneurones with axons that ramified extensively in the spinal cord, including the IML and other autonomic regions. In contrast, all cells that were insensitive to TEA and 4-AP were confirmed as sympathetic preganglionic neurones. Both electrophysiological and morphological data are therefore consistent with the presence of the voltage-gated potassium channel subunit Kv3.1 in interneurones, but not sympathetic preganglionic neurones. Testing this proposal immunohistochemically revealed that Kv3.1b was localised in low numbers of neurones within the IML but in higher numbers of neurones on the periphery of the IML. Kv3.1b-expressing neurones were not sympathetic preganglionic neurones since they were not retrogradely labelled following intraperitoneal injections of Fluorogold. Since Kv3.2 plays a similar role to Kv3.1 we also tested for the presence of Kv3.2 using immunohistochemistry, but failed to detect it in neuronal somata in the spinal cord. These studies provide electrophysiological and morphological data on interneurones in the IML and indicate that the channels containing the Kv3.1 subunit are important in setting the firing pattern of these neurones.     

Immunohistochemical localization of the voltage-gated potassium channel subunit Kv1.4 in the central nervous system of the adult rat

A large set of voltage-gated potassium channels is involved in regulating essential aspects of neuronal function in the central nervous system, thus contributing to the ability of neurons to respond to a given input. In the present study, we used immunocytochemical methods to elucidate the regional, cellular and subcellular distribution of the voltage-gated potassium channel subunit Kv1.4, a member of the Shaker subfamily, in the brain. At the light microscopic level, the Kv1.4 subunit showed a unique distribution pattern, being localized in specific neuronal populations of the rat brain. The neuronal regions expressing the highest levels of Kv1.4 protein included the cerebral cortex, the hippocampus, the posterolateral and posteromedial ventral thalamic nuclei, the dorsolateral and medial geniculate nuclei, the substantia nigra and the dorsal cochlear nucleus. The Kv1.4 subunit was also present in other neuronal populations, with different levels of Kv1.4 immunoreactivity. In all immunolabeled regions, the Kv1.4 subunit was mostly diffusely distributed and, to a lesser extent, it stained cell bodies and proximal dendrites. Furthermore, Kv1.4 immunoreactivity was also detected in nerve terminals and axonal terminal fields. At the electron microscopic level, Kv1.4 was located postsynaptically in dendritic spines and shafts at extrasynaptic sites, as well as presynaptically in axon and active zone of axon terminals, in the neocortex and hippocampus. The findings indicate that Kv1.4 channels are widely distributed in the rat brain and suggest that activation of this channel would have different modulatory effects on neuronal excitability.     

Molecular basis for different pore properties of potassium channels from the rat brain Kv1 gene family

 Members of the rat brain Kv1 family of cloned potassium channels are structurally highly homologous, but have diverse conductance and pharmacological characteristics. Here we present data on the effects of mutating residues K533 in the P-region and H471 in the S4-S5 linker of Kv1.4 to their equivalent residues in Kv1.1 and Kv1.6 on single-channel conductance and sensitivity to external tetraethylammonium cations (TEA⁺) and internal Mg²⁺. Exchange of residue K533 for its equivalent residue (Y) in Kv1.1 and Kv1.6 increased the single-channel conductance at both negative and positive potentials. This mutation is known to reduce the IC₅₀ for external TEA⁺ from > 100 mM to 0.6 mM, almost identical to that for Kv1.1 (0.53 mM). We have now found that the additional exchange of residue H471 for the equivalent residue (K) in Kv1.6 increased the IC₅₀ for external TEA⁺ from 0.6 mM (Kv1.4K533Y) to 2.39 mM; this is very close to that for wild-type Kv1.6 channels (2.84 mM). The mutation H471K alone was ineffective. We thus provide evidence that the S4-S5 linker does contribute to the channel’s inner-pore region. Data on the block of Kv1 channels by internal Mg²⁺ indicate that while the binding site is probably situated within the deep-pore region, its exact location may be channel specific. Received: 25 February 1997 / Received after revision: 4 June 1997 / Accepted: 10 June 1997     

Expression and distribution of Kv4 potassium channel subunits and potassium channel interacting proteins in subpopulations of interneurons in the basolateral amygdala

The Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary β-subunits, such as the potassium channel interacting proteins (KChIP1 - 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other β-subunits (KChIP2-4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and β-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immunoreactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA.     

Cysteine-modifying reagents alter the gating of the rat cloned potassium channel Kv1.4

The effects of cysteine-modifying reagents on the gating of rat cloned Kv1.4 channels expressed in HEK-293 cells were examined using the whole-cell patch-clamp technique. Cells transfected with Kv1.4 expressed a rapidly inactivating K⁺ current with a midpoint of activation of –31 mV and a slope factor of 5 mV measured with tail current protocols in 35 mM Rb⁺ external solutions. The cysteine-specific oxidizing agents 2,2-dithiobis-5-nitropyridine (DTBNP, 50 M) and chloramine-T (CL-T, 500 M) removed inactivation of Kv1.4. These effects were reversed by the reducing agent dithiothreitol (DTT, 10 mM). In addition, DTBNP and CL-T also slowed Kv1.4 deactivation and increased the voltage sensitivity of deactivation. The action of cysteine-modifying reagents on Kv1.4 suggests that redox state affects channel gating, with oxidation tending to stabilize the open state of the channel, both by removing inactivation and slowing deactivation.     

Membrane cholesterol modulates Kv1.5 potassium channel distribution and function in rat cardiomyocytes

Membrane lipid composition is a major determinant of cell excitability. In this study, we assessed the role of membrane cholesterol composition in the distribution and function of Kv1.5-based channels in rat cardiac membranes. In isolated rat atrial myocytes, the application of methyl-β-cyclodextrin (MCD), an agent that depletes membrane cholesterol, caused a delayed increase in the Kv1.5-based sustained component, I _kur, which reached steady state in ∼7 min. This effect was prevented by preloading the MCD with cholesterol. MCD-increased current was inhibited by low 4-aminopyridine concentration. Neonatal rat cardiomyocytes transfected with Green Fluorescent Protein (GFP)-tagged Kv1.5 channels showed a large ultrarapid delayed-rectifier current ( I _Kur), which was also stimulated by MCD. In atrial cryosections, Kv1.5 channels were mainly located at the intercalated disc, whereas caveolin-3 predominated at the cell periphery. A small portion of Kv1.5 floated in the low-density fractions of step sucrose-gradient preparations. In live neonatal cardiomyocytes, GFP-tagged Kv1.5 channels were predominantly organized in clusters at the basal plasma membrane. MCD caused reorganization of Kv1.5 subunits into larger clusters that redistributed throughout the plasma membrane. The MCD effect on clusters was sizable 7 min after its application. We conclude that Kv1.5 subunits are concentrated in cholesterol-enriched membrane microdomains distinct from caveolae, and that redistribution of Kv1.5 subunits by depletion of membrane cholesterol increases their current-carrying capacity.     

Kv3.1b and Kv3.3 channel subunit expression in murine spinal dorsal horn GABAergic interneurones

GABAergic interneurones, including those within spinal dorsal horn, contain one of the two isoforms of the synthesizing enzyme glutamate decarboxylase (GAD), either GAD65 or GAD67. The physiological significance of these two GABAergic phenotypes is unknown but a more detailed anatomical and functional characterization may help resolve this issue. In this study, two transgenic Green Fluorescent Protein (GFP) knock-in murine lines, namely GAD65-GFP and GAD67-GFP (Δneo) mice, were used to profile expression of Shaw-related Kv3.1b and Kv3.3 K⁺-channel subunits in dorsal horn interneurones. Neuronal expression of these subunits confers specific biophysical characteristic referred to as ‘fast-spiking’. Immuno-labelling for Kv3.1b or Kv3.3 revealed the presence of both of these subunits across the dorsal horn, most abundantly in laminae I-III. Co-localization studies in transgenic mice indicated that Kv3.1b but not Kv3.3 was associated with GAD65-GFP and GAD67-GFP immunopositive neurones. For comparison the distributions of Kv4.2 and Kv4.3 K⁺-channel subunits which are linked to an excitatory neuronal phenotype were characterized. No co-localization was found between GAD-GFP +ve neurones and Kv4.2 or Kv4.3. In functional studies to evaluate whether either GABAergic population is activated by noxious stimulation, hindpaw intradermal injection of capsaicin followed by c-fos quantification in dorsal horn revealed co-expression c-fos and GAD65-GFP (quantified as 20-30% of GFP +ve population). Co-expression was also detected for GAD67-GFP +ve neurones and capsaicin-induced c-fos but at a much reduced level of 4-5%. These data suggest that whilst both GAD65-GFP and GAD67-GFP +ve neurones express Kv3.1b and therefore may share certain biophysical traits, their responses to peripheral noxious stimulation are distinct.     

Neuronal activity and TrkB ligands influence Kv3.1b and Kv3.2 expression in developing cortical interneurons

Among the GABAergic neocortical interneurons, fast-spiking (FS) basket and chandelier cells are essential mediators for feed-forward inhibition, network synchrony and oscillations. The FS properties are in part mediated by the voltage-gated potassium channels Kv3.1b/3.2 which allow the fast repolarization of the membrane necessary for firing non-adapting action potentials at high frequencies. It has been recently reported that the FS phenotype fails to mature in BDNF knockout mice suggesting a role for neurotrophins. We now describe the role of neuronal activity and neurotrophins for Kv3.1b/3.2 expression using organotypic cultures of rat visual cortex as model system. Chronic activity deprivation from 2 days in vitro (DIV) prevented the postnatal developmental increase of Kv3.2, but not Kv3.1b mRNA expression. However, chronic activity deprivation failed to alter Kv3.1b and marginally delayed Kv3.2 protein expression. Activity deprivation by glutamate receptor blockade from 10 to 20 DIV reduced both mRNAs, whereas deprivation with tetrodotoxin (TTX) reduced both mRNAs and the Kv3.2 protein. Thalamic and cortical afferents in cocultures failed to alter the expression. BDNF and NT4 supplemented from 2 DIV onwards increased the expression of Kv3.1b, but not Kv3.2 mRNA in young cultures. Only NT4 increased the expression of both mRNAs later in development. Kv3 protein levels were not changed by exogenous tropomyosin-related kinase B (TrkB) ligands, but the levels decreased upon inhibiting the MAPK signaling suggesting a role for endogenous factors and in particular MEK2 signaling for translation. The results show that Kv3.1b/3.2 expression is differentially controlled by neuronal activity and neurotrophic factors.     

Manipulation of the potassium channel Kv1.1 and its effect on neuronal excitability in rat sensory neurons

Potassium channels play a critical role in regulating many aspects of action potential (AP) firing. To establish the contribution of the voltage-dependent potassium channel Kv1.1 in regulating excitability, we used the selective blocker dendrotoxin-K (DTX-K) and small interfering RNA (siRNA) targeted to Kv1.1 to determine their effects on AP firing in small-diameter capsaicin-sensitive sensory neurons. A 5-min exposure to 10 nM DTX-K suppressed the total potassium current (I(K)) measured at +40 mV by about 33%. DTX-K produced a twofold increase in the number of APs evoked by a ramp of depolarizing current. Associated with increased firing was a decrease in firing threshold and rheobase. DTX-K did not alter the resting membrane potential or the AP duration. A 48-h treatment with siRNA targeted to Kv1.1 reduced the expression of this channel protein by about 60% as measured in Western blots. After treatment with siRNA, I(K) was no longer sensitive to DTX-K, indicating a loss of functional protein. Similarly, after siRNA treatment exposure to DTX-K had no effect on the number of evoked APs, firing threshold, or rheobase. However, after siRNA treatment, the firing threshold had values similar to those obtained after acute exposure to DTX-K, suggesting that the loss of Kv1.1 plays a critical role in setting this parameter of excitability. These results demonstrate that Kv1.1 plays an important role in limiting AP firing and that siRNA may be a useful approach to establish the role of specific ion channels in the absence of selective antagonists.