The molecular biology of K+ channels.

It is now clear that voltage-gated K+ channels are encoded by a set of multigene subfamilies. Expression of different members of these subfamilies, coupled with mutational analysis, has advanced our knowledge of the structure and function of voltage-dependent K+ channels.

Expression of the Kv3.1 potassium channel in the avian auditory brainstem.

The Shaw-like potassium channel Kv3.1, a delayed rectifier with a high threshold of activation, is expressed in the time coding nuclei of the bird auditory brainstem. In both barn owls and chickens, Kv3.1 mRNA was expressed in the cochlear nucleus magnocellularis (NM) and the nucleus laminaris (NL). Western blot analysis showed that an antibody raised against the synthetic peptide sequence of rat Kv3.1 (rKv3.1) specifically recognized the same 92 kDa protein bands in both rat and chicken synaptosomal preparations. Immunohistochemical analyses using this anti-rKv3.1 antibody revealed a prominent gradient in Kv3.1 immunoreactivity along the tonotopic axis of the barn owl NM and NL and a less prominent gradient in the chicken. The precise localization of the Kv3.1 immunoproduct was resolved by electron microscopy. In both the owl and the chicken, Kv3.1 was targeted postsynaptically in NM and NL. The major difference in localization of Kv3.1 protein between the two birds was the expression of Kv3.1 in the NM axons and terminals in the region of the barn owl NL. This location of Kv3.1 channels supports its postulated function in reducing the width of action potentials as they invade the presynaptic terminal. The presynaptic localization may be a specialization for enabling neurons in owl NM to transmit high-frequency temporal information with little jitter.

Localization of a high threshold potassium channel in the rat cochlear nucleus.

Potassium channels play a major role in determining the pattern and frequency of neuronal firing. In the cochlear nucleus (CN), various morphologically defined types of neurons have different responses to a sound. We have previously identified one type of cloned K+ channel, termed Kv3.1, which is highly expressed in many auditory neurons. Expression studies indicate that Kv3.1 channels have an unusually high threshold for activation. In this study, we used both in situ hybridization and immunohistochemistry to examine the expression patterns of the Kv3.1 channel in the CN. In the ventral CN, bushy cells hybridized strongly with Kv3.1 specific probes and a subpopulation of stellate/multipolar cells hybridized with Kv3.1 probes. In the dorsal CN, pyramidal and large multipolar/giant cells expressed Kv3.1 mRNA. Abundant Kv3.1 immunolabeling was also observed in the CN. The pattern of immunolabeling revealed that the Kv3.1 protein is distributed along the soma, proximal dendrites, unmyelinated axons, and axon terminals of stained neurons. In the case of pyramidal and octopus cells, no immunolabeling was detected at the somata, even though these cells expressed Kv3.1 mRNA. Computer simulations were used to explore the functional role of the Kv3.1 channel. The simulations indicate that Kv3.1 conductances may contribute to repolarization of large synaptic potentials. When stimulated at high frequencies, the presence of Kv3.1 enhances the ability of a model cell with some of the features of bushy cells to follow high frequency input with temporal precision.

Localization of two high-threshold potassium channel subunits in the rat central auditory system.

The firing pattern of auditory neurons is determined in part by the type of voltage-sensitive potassium channels expressed. The expression patterns for two high-threshold potassium channels, Kv3.1 and Kv3.3, that differ in inactivation properties were examined in the rat auditory system. The positive activation voltage and rapid deactivation kinetics of these channels provide rapid repolarization of action potentials with little effect on action potential threshold. In situ hybridization experiments showed that Kv3.3 mRNA was highly expressed in most auditory neurons in the rat brainstem, whereas Kv3.1 was expressed in a more limited population of auditory neurons. Notably, Kv3.1 mRNA was not expressed in neurons of the medial and lateral superior olive and a subpopulation of neurons in the ventral nucleus of the lateral lemniscus. These results suggest that Kv3.3 channels may be the dominant Kv3 subfamily member expressed in brainstem auditory neurons and that, in some auditory neurons, Kv3.1 and Kv3.3 may coassemble to form functional channels. The localization of Kv3.1 protein was examined immunohistochemically. The distribution of stained somata and neuropil varied across auditory nuclei and correlated with the distribution of Kv3.1 mRNA-expressing neurons and their terminal arborizations, respectively. The intensity of Kv3.1 immunoreactivity varied across the tonotopic map in the medial nucleus of the trapezoid body with neurons responding best to high-frequency tones most intensely labeled. Thus, auditory neurons may vary the types and amount of K(+) channel expression in response to synaptic input to subtly tune their firing properties.

Depolarization-induced increases in intracellular free calcium detected in single cultured neuronal cells.

Microspectrofluorometry was used to examine intracellular free calcium changes in single NG108-15 neurons loaded with the Ca2+ sensitive probe, quin2. The changes in intracellular free Ca2+ were induced either by depolarizing cells with high K+ or veratridine or by the addition of ionomycin. Ca2+-free medium and various inorganic and organic calcium-channel blocking agents blocked changes in intracellular free Ca2+ levels, indicating that Ca2+ entry is most likely through voltage-sensitive calcium channels.

Two different G-proteins mediate neuropeptide Y and bradykinin-stimulated phospholipid breakdown in cultured rat sensory neurons.

We have previously demonstrated that neuropeptide Y (NPY) inhibits voltage sensitive Ca2+ channels in rat dorsal root ganglion neurons and that this effect is mediated by a pertussis toxin-sensitive, guanyl nucleotide-binding protein (G-protein). We now demonstrate that NPY can also stimulate the synthesis of inositol trisphosphate (InsP3) and diacylglycerol in dorsal root ganglion neurons. The effects of NPY were compared with those of bradykinin (BK) which also stimulates phosphoinositide turnover in these cells. NPY-stimulated InsP3 synthesis could be completely blocked by treatment with pertussis toxin and significantly enhanced by cholera toxin although not by other agents which raised cellular concentrations of cyclic AMP. In contrast, the effects of BK were completely unaltered by either toxin. Furthermore the maximal effects of BK and NPY were additive. In spite of the lack of toxin effects, stimulation of InsP3 synthesis produced by BK was clearly mediated by a G-protein. Thus BK stimulated InsP3 production in digitonin-permeabilized neurons, and these effects were enhanced by guanosine 5'-O-(3-thiotriphosphate) and blocked by guanosine 5'-O-(2-thiodiphosphate). The stimulatory effects of both NPY and BK were also blocked by treatment of neurons with phorbol esters. Fura-2-based microfluorimetry of single dorsal root ganglion neurons demonstrated that both BK and NPY increased cytoplasmic-free Ca2+ concentration and that both peptides could produce this effect in the same neuron. Both agents could still increase cytoplasmic-free Ca2+ concentration in Ca2+-free medium indicating that the source of the Ca2+ was an intracellular store. Thus, both NPY and BK can activate InsP3 synthesis in the same cell but seem to utilize different G-proteins. NPY utilizes a pertussis toxin-sensitive G-protein and BK a toxin-insensitive one.

Regulation of calcium homeostasis in sensory neurons by bradykinin.

The nonapeptide bradykinin (BK) activates sensory neurons and stimulates the transmission of nociceptive information into the CNS. We investigated the effect of this peptide on rat dorsal root ganglion neurons (DRG) grown in vitro. BK stimulated the synthesis of inositol trisphosphate (IP3) and the breakdown of phosphatidylinositol bisphosphate, the synthesis of diacylglycerol, and the release of arachidonic acid from DRG cells. The release of IP3 and arachidonic acid was not inhibited by pretreatment of the cells with pertussis toxin. BK also mobilized intracellular Ca2+ stores in DRG cells as assessed by fura-2-based microfluorimetry. Two types of Ca2+ stores appeared to exist in DRG neurons. One type could be mobilized by caffeine (10(-2) M), and this effect could be blocked by ryanodine in a use-dependent manner. These stores occurred primarily in the cell soma and were virtually absent from cell processes. A second type of store could be mobilized by BK, presumably through the mediation of IP3. These latter stores were distributed equally between the cell soma and processes. Experiments with combinations of caffeine and BK suggested that the stores mobilized by these 2 agents may be separate entities. Both the caffeine and BK sensitive Ca2+ storage sites appeared to participate in buffering a Ca2+ load induced in DRG neurons by cell depolarization. The relevance of these observations to the mechanism of action of BK on sensory neurons is discussed.

Cloning and characterization of the promoter for a potassium channel expressed in high frequency firing neurons.

The Kv3.1 potassium channel is expressed in neurons that generate trains of high frequency action potentials in response to synaptic inputs. To understand the mechanisms underlying the regulation and restricted expression pattern of the Kv3.1 gene, we have cloned and characterized its promoter. We first isolated a 5.3-kilobase pair fragment of the Kv3.1 5'-flanking region. When linked to the chloramphenicol acetyltransferase reporter gene, this fragment was found to be active in the undifferentiated PC12 cell line, a neuron-like cell line, but not in a fibroblast cell line. By carrying out a series of deletion analyses in undifferentiated PC12 cells, we have localized the essential promoter region to a highly GC-rich region containing four Sp-1 binding sites. Similar deletion analysis in NIH3T3 cells suggests that multiple silencing elements and enhancing element(s) are involved in the cell type-specific expression of this gene. Further regulatory elements, including one cyclic AMP/calcium response element (CRE) and one Ap-1 element were found in the upstream region of the promoter. Using a stable undifferentiated PC12 cell line transfected with the Kv3.1 5'-flanking region, we determined that promoter activity is enhanced by a cAMP analog and a calcium ionophore. Deletion of the CRE-like element at position -252 eliminated the enhancement of promoter activity by cAMP, and mobility shift assays confirmed that the Kv3.1 CRE sequence binds both a nuclear factor in undifferentiated PC12 cells and recombinant CRE binding protein. Our results suggest that the transcription of the Kv3.1 channel may be regulated by neurotransmitters that elevate cAMP levels in neurons.

Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons.

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high-affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.

Expression of the mRNAs for the Kv3.1 potassium channel gene in the adult and developing rat brain.

1. The gene for a mammalian Shaw K+ channel has recently been cloned and has been shown, by alternative splicing, to give rise to two different transcripts, Kv3.1 alpha and Kv3.1 beta. To determine whether these channels are associated with specific types of neurons and to determine whether or not the alternately spliced K+ channel variants are differentially expressed, we used ribonuclease (RNase) protection assays and in situ hybridization histochemistry to localize the specific subsets of neurons containing Kv3.1 alpha and Kv3.1 beta mRNAs in the adult and developing rat brain. 2. In situ hybridization histochemistry revealed a heterogeneous expression pattern of Kv3.1 alpha mRNA in the adult rat brain. Highest Kv3.1 alpha mRNA levels were expressed in the cerebellum. High levels of hybridization were also detected in the globus pallidus, subthalamus, and substantia nigra reticulata. Many thalamic nuclei, but in particular the reticular thalamic nucleus, hybridized well to Kv3.1 alpha-specific probes. A subpopulation of cells in the cortex and hippocampus, which by their distribution and number may represent interneurons, were also found to contain high levels of Kv3.1 alpha mRNA. In the brain stem, many nuclei, including the inferior colliculus and the cochlear and vestibular nuclei, also express Kv3.1 alpha mRNA. Low or undetectable levels of Kv3.1 alpha mRNA were found in the caudate-putamen, olfactory tubercle, amygdala, and hypothalamus. 3. Kv3.1 beta mRNA was also detected in the adult rat brain by both RNase protection assays and by in situ hybridization experiments. Although the beta splice variant is expressed at lower levels than the alpha species, the overall expression pattern for both mRNAs is similar, indicating that both splice variants co-expressed in the same neurons. 4. The expression of Kv3.1 alpha and Kv3.1 beta transcripts was examined throughout development. Kv3.1 alpha mRNA is detected as early as embryonic day 17 and then increases gradually until approximately postnatal day 10, when there is a large increase in the amount of Kv3.1 alpha mRNA. Interestingly, the expression of Kv3.1 beta mRNA only increases gradually during the developmental time frame examined. Densitometric measurements indicated that Kv3.1 alpha is the predominant splice variant found in neurons of the adult brain, whereas Kv3.1 beta appears to be the predominant species in embryonic and perinatal neurons. 5. Most of the neurons that express the Kv3.1 transcripts have been characterized electrophysiologically to have narrow action potentials and display high-frequency firing rates with little or no spike adaptation.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple calcium channels mediate neurotransmitter release from peripheral neurons.

We examined the effects of dihydropyridine drugs on evoked neurotransmitter release from cultured neonatal rat sensory and sympathetic neurons. Depolarization with K+-rich solutions increased the release of substance P from cultured sensory neurons. This release was enhanced by BAY K8644 and (+)-202791 and was blocked by a variety of other dihydropyridines including (-)-202791, by Co2+, or in Ca2+-free solutions. K+-rich solutions also stimulated the release of [3H]norepinephrine from cultured sympathetic neurons. This release was also completely blocked by Co2+ or in Ca2+-free solution. In contrast to the situation in sensory neurons, however, the evoked release of [3H]norepinephrine was completely resistant to the blocking effects of dihydropyridine such as nimodipine. However, BAY K8644 was able to enhance the evoked release of [3H]norepinephrine, and this enhancement was blocked by nimodipine. These results are discussed in relation to the possible participation of multiple types of calcium channels in the release of neurotransmitters.

GABA-ergic interneurons of the striatum express the Shaw-like potassium channel Kv3.1.

In addition to numerous GABA-ergic efferent neurons, the striatum contains a subpopulation of fast-firing GABA-ergic interneurons characterized by the presence of immunoreactivity for the calcium-binding protein, parvalbumin. Double-label in situ hybridization with digoxigenin- and radiolabelled cRNA probes was performed on striatal sections of adult rats to identify mRNAs expressed by striatal GABA-ergic interneurons. In the dorsolateral striatum, only parvalbumin mRNA-positive neurons expressed the mRNA encoding the potassium channel Kv3.1, a member of the Shaw family of potassium channels with rapid activation and inactivation kinetics, usually found in fast-firing neurons such as the basket cells of the hippocampus. Colocalization of the parvalbumin and Kv3.1 proteins was confirmed by double-label immunohistochemistry. Parvalbumin mRNA-positive neurons expressed very high levels of the mRNA encoding glutamic acid decarboxylase (Mr 67,000: GAD67) in the dorsolateral striatum. A smaller proportion of double-labelled neurons was found in the ventrolateral striatum. A small number of densely labelled neurons for GAD67 mRNA also expressed the mRNA encoding the dopamine D2 receptor, but none expressed detectable levels of the dopamine D1 receptor mRNA. This indicates major differences in the expression of dopamine receptor mRNA in a majority of GABA-ergic interneurons vs. GABA-ergic efferent neurons of the striatum. The results suggest that distinct molecular characteristics are associated with the distinct electrophysiological properties of striatal GABA-ergic neurons.

Electrophysiological and pharmacological characterization of a mammalian Shaw channel expressed in NIH 3T3 fibroblasts.

1. The Shaw-like voltage-activated potassium channel Kv3.1 is expressed in neurons that generate rapid trains of action potentials. By expressing this channel in a mammalian cell line and by simulating its activation, we tested the potential role of this channel in action potential repolarization. 2. NIH 3T3 fibroblasts were stably transfected with Kv3.1 DNA. Currents recorded in these cells had a threshold of activation at approximately -10 mV, showed little inactivation, and were very sensitive to blockade by 4-aminopyridine and tetraethylammonium. 3. Kv3.1 currents activated rapidly at the onset of depolarizing voltage pulses. After an initial rapid phase of activation, which could be fit by an n4 Hodgkin-Huxley model, Kv3.1 currents expressed in fibroblasts had a second, slower phase of activation, and, in some cells, a slower phase of partial inactivation, both of which could be fit with modified n4p models. 4. Cell-attached single-channel recordings indicated that the Kv3.1 channel displays two gating behaviors, a short-open-time pattern, which occurs only at the onset of depolarization, and a long-open-time pattern, which predominates during prolonged depolarizations. 5. The amplitude of Kv3.1 currents, and the probability of channel openings, was reduced by a phorbol ester activator of protein kinase C, and the action of this agent was blocked by preincubation with the protein kinase inhibitor H7 (1-[5-isoquinolinesulfonyl]-2-methyl piperazine). In contrast, the effects of dioctanoyl glycerol, which also attenuated the currents, could not be completely blocked by H7, suggesting that diacylglycerols may act on the channel by a kinase-independent pathway. 6. Incorporation of a current with the kinetics and voltage dependence of Kv3.1 currents into a model cell with a sustained inward current showed that, in contrast to other delayed-rectifier currents such as the Shaker-like Kv1.1 and Kv1.6 channels, the level of expression of Kv3.1 currents could be varied over a wide range without attenuation of action potential height. Our results suggest that the Kv3.1 channel may provide rapidly firing neurons with a high safety factor for impulse propagation.

Activation of Kv3.1 channels in neuronal spine-like structures may induce local potassium ion depletion.

Spines are specialized neuronal membrane structures, often localized at sites where synaptic information is relayed from one cell to another in the central nervous system. By electron immunomicroscopy we have found that the mammalian Shaw family potassium channel Kv3.1 is localized on spine-like protrusions, adjacent to postsynaptic membranes of bushy cells in the cochlear nucleus. As direct characterization of the electrophysiological behavior of ion channels in such structures is difficult, we have used Kv3. 1-transfected CHO cells to create artificial spine-like membrane compartments. Membrane patches were sucked into microelectrodes to form small, cell-attached vesicles with dimensions comparable to those of the neuronal structures. Currents mediated by the Kv3.1 channel in these vesicles undergo rapid and complete inactivation, in contrast to their noninactivating behavior in whole-cell recordings. This apparent inactivation is caused by the rapid depletion of K+ from the vesicle and the slow refilling of K+ into the vesicle compartment from the bulk cytoplasm. Our data provide evidence that compartmentalized ionic transients can be generated in spine-like membrane structures and support the view that the localization of ion channels in spine-like structures may influence responses to synaptic stimulation.

The effect of down regulation of protein kinase C on the inhibitory modulation of dorsal root ganglion neuron Ca2+ currents by neuropeptide Y.

Dorsal root ganglion (DRG) neurons cultured from neonatal rats contained high concentrations of protein kinase C (PKC). Normally, the majority of the enzyme activity was found in the cytosol and considerably less was associated with the membrane fraction. Upon incubation with the phorbol ester phorbol dibutyrate (PDBu, 10(-6) M) for 20 min, PKC activity increased in the membrane-associated fraction and decreased in the cytoplasmic fraction. Longer incubations with phorbol ester also induced a decline in membrane-associated PKC activity. If incubations were continued for periods of over 10 hr, both membrane and cytosolic PKC activity declined essentially to zero. Down-regulation of PKC had no effect on the number or affinity of 125I-neuropeptide Y (NPY) binding sites on DRG cells or on the absolute magnitude of the DRG Ca2+ current. However, the ability of NPY to inhibit the DRG Ca2+ current was greatly reduced. When sustained Ca2+ currents were evoked from depolarized holding potentials (-40 mV), all concentrations of NPY (10(-10)-10(-7) M) were less effective. In contrast, higher concentrations of NPY still blocked the transient portion of the DRG Ca2+ current evoked from hyperpolarized holding potentials. These results support the suggestion that PKC is involved in the inhibitory modulation of DRG Ca2+ currents by neurotransmitters. The precise role of PKC may vary depending on the type of Ca2+ channel involved.

Expression of the H-ras oncogene induces potassium conductance and neuron-specific potassium channel mRNAs in the AtT20 cell line.

Expression of the EJ-ras oncogene in the AtT20 cell line results in several changes in their properties that correspond to a switch of these anterior pituitary-derived cells to a more neuronlike phenotype. The width of action potentials following transfection with ras is reduced 20-fold from over 200 msec in control AtT20 cells to less than 10 msec in ras-transfected cells. This is associated with a two- to threefold increase in the density of voltage-dependent potassium currents. In addition, the rate of inactivation of these currents is decreased approximately twofold in ras-transfected cells. At least part of the change in potassium current may be due to differential expression of potassium channel mRNAs. In the ras-transfected cells, mRNA species were detected using a probe for the voltage-dependent potassium channels, Kv4, a species that appears to be uniquely expressed in the nervous system, and NGK2, an alternatively spliced product transcribed from the same gene. These mRNAs are not detected in control AtT20 cells. The results suggest that the ras protein modulates the phenotype of excitable cells by influencing the expression of specific potassium channels and thereby altering the density and types of channels in the plasma membrane.