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.

Mitochondrial factors with dual roles in death and survival.

At least in mammals, we have some understanding of how caspases facilitate mitochondria-mediated cell death, but the biochemical mechanisms by which other factors promote or inhibit programmed cell death are not understood. Moreover, most of these factors are only studied after treating cells with a death stimulus. A growing body of new evidence suggests that cell death regulators also have 'day jobs' in healthy cells. Even caspases, mitochondrial fission proteins and pro-death Bcl-2 family proteins appear to have normal cellular functions that promote cell survival. Here, we review some of the supporting evidence and stretch beyond the evidence to seek an understanding of the remaining questions.

Kv1.3 channel gene-targeted deletion produces "Super-Smeller Mice" with altered glomeruli, interacting scaffolding proteins, and biophysics.

Mice with gene-targeted deletion of the Kv1.3 channel were generated to study its role in olfactory function. Potassium currents in olfactory bulb mitral cells from Kv1.3 null mice have slow inactivation kinetics, a modified voltage dependence, and a dampened C-type inactivation and fail to be modulated by activators of receptor tyrosine signaling cascades. Kv1.3 deletion increases expression of scaffolding proteins that normally regulate the channel through protein-protein interactions. Kv1.3-/- mice have a 1,000- to 10,000-fold lower threshold for detection of odors and an increased ability to discriminate between odorants. In accordance with this heightened sense of smell, Kv1.3-/- mice have glomeruli or olfactory coding units that are smaller and more numerous than those of wild-type mice. These data suggest that Kv1.3 plays a far more reaching role in signal transduction, development, and olfactory coding than that of the classically defined role of a potassium channel-to shape excitability by influencing membrane potential.

Targeted attenuation of electrical activity in Drosophila using a genetically modified K(+) channel.

We describe here a general technique for the graded inhibition of cellular excitability in vivo. Inhibition is accomplished by expressing a genetically modified Shaker K(+) channel (termed the EKO channel) in targeted cells. Unlike native K(+) channels, the EKO channel strongly shunts depolarizing current: activating at potentials near E(K) and not inactivating. Selective targeting of the channel to neurons, muscles, and photoreceptors in Drosophila using the Gal4-UAS system results in physiological and behavioral effects consistent with attenuated excitability in the targeted cells, often with loss of neuronal function at higher transgene dosages. By permitting the incremental reduction of electrical activity, the EKO technique can be used to address a wide range of questions regarding neuronal function.

Presynaptic target of Ca2+ action on neuropeptide and acetylcholine release in Aplysia californica.

1. When buccal neuron B2 of Aplysia californica is co-cultured with sensory neurons (SNs), slow peptidergic synapses are formed. When B2 is co-cultured with neurons B3 or B6, fast cholinergic synapses are formed. 2. Patch pipettes were used to voltage clamp pre- and postsynaptic neurons and to load the caged Ca2+ chelator o-nitrophenyl EGTA (NPE) and the Ca2+ indicator BTC into presynaptic neurons. The relationships between presynaptic [Ca2+]i and postsynaptic responses were compared between peptidergic and cholinergic synapses formed by cell B2. 3. Using variable intensity flashes, Ca2+ stoichiometries of peptide and acetylcholine (ACh) release were approximately 2 and 3, respectively. The difference did not reach statistical significance. 4. ACh quanta summate linearly postsynaptically. We also found a linear dose-response curve for peptide action, indicating a linear relationship between submaximal peptide concentration and response of the SN. 5. The minimum intracellular calcium concentrations ([Ca2+]i) for triggering peptidergic and cholinergic transmission were estimated to be about 5 and 10 microM, respectively. 6. By comparing normal postsynaptic responses to those evoked by photolysis of NPE, we estimate [Ca2+]i at the release trigger site elicited by a single action potential (AP) to be at least 10 microM for peptidergic synapses and probably higher for cholinergic synapses. 7. Cholinergic release is brief (half-width approximately 200 ms), even in response to a prolonged rise in [Ca2+]i, while some peptidergic release appears to persist for as long as [Ca2+]i remains elevated (for up to 10 s). This may reflect differences in sizes of reserve pools, or in replenishment rates of immediately releasable pools of vesicles. 8. Electron microscopy revealed that most synaptic contacts had at least one morphologically docked dense core vesicle that presumably contained peptide; these were often located within conventional active zones. 9. Both cholinergic and peptidergic vesicles are docked within active zones, but cholinergic vesicles may be located closer to Ca2+ channels than are peptidergic vesicles.

Calmodulin regulates assembly and trafficking of SK4/IK1 Ca2+-activated K+ channels.

Calmodulin (CaM) regulates gating of several types of ion channels but has not been implicated in channel assembly or trafficking. For the SK4/IK1 K+ channel, CaM bound to the proximal C terminus ("Ct1 " domain) acts as the Ca2+ sensor. We now show that CaM interacting with the C terminus of SK4 also controls channel assembly and surface expression. In transfected cells, removing free CaM by overexpressing the CaM-binding domain, Ct1, redistributed full-length SK4 protein from the plasma membrane to the cytoplasm and decreased whole-cell currents. Making more CaM protein available by overexpressing the CaM gene abrogated the dominant-negative effect of Ct1 and restored both surface expression of SK4 protein and whole-cell currents. The distal C-terminal domain ("Ct2") also plays a role in assembly, but is not CaM-dependent. Co-immunoprecipitation experiments demonstrated that multimerization of SK4 subunits was enhanced by CaM and inhibited by removal of CaM, indicating that CaM regulates trafficking of SK4 by affecting the assembly of channels. Our results support a model in which CaM-dependent association of SK4 monomers at their Ct1 domains regulates channel assembly and surface expression. This appears to represent a novel mechanism for controlling ion channels, and consequently, the cellular functions that depend on them.

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.

Aplysia ror forms clusters on the surface of identified neuroendocrine cells.

The ror receptors are a highly conserved family of receptor tyrosine kinases genetically implicated in the establishment of cellular polarity. We have cloned a ror receptor from the marine mollusk Aplysia californica. Aplysia ror (Apror) is expressed in most developing neurons and some adult neuronal populations, including the neuroendocrine bag-cell neurons. The Apror protein is present in peripheral neuronal processes and ganglionic neuropil, implicating the kinase in the regulation of growth and/or synaptic events. In cultured bag-cell neurons, the majority of the protein is stored in intracellular organelles, whereas only a small fraction is expressed on the surface. When expressed on the cell surface, the protein is clustered on neurites, suggesting that Apror is involved in the organization of functional domains within neurons. Apror immunoreactivity partially colocalizes with the P-type calcium channel BC-alpha1A at bag-cell neuron varicosities, suggesting a role for Apror in organizing neuropeptide release sites.

Casein kinase 2 determines the voltage dependence of the Kv3.1 channel in auditory neurons and transfected cells.

The Kv3.1 potassium channel can be distinguished from most other delayed rectifier channels by its very high threshold of activation and lack of use-dependent inactivation. This allows neurons that express this channel to fire at very high frequencies. We have now found that this feature of the Kv3.1 channel is strongly influenced by its constitutive phosphorylation by the enzyme casein kinase II. Using stably transfected Chinese hamster ovary cells expressing Kv3.1, we show that Kv3.1 is highly phosphorylated under basal conditions. Whole-cell patch clamp recordings were used to characterize the electrophysiological consequence of dephosphorylation using alkaline phosphatase. This enzyme produced an increase in whole-cell conductance and shifted the voltage dependence of activation to more negative potentials by >20 mV. In addition, a similar shift in the voltage dependence of inactivation was observed. These findings were also confirmed in native Kv3.1 channels expressed in medial nucleus of the trapezoid body (MNTB) neurons. Furthermore, inhibitors of casein kinase 2 mimicked the effect of phosphatase treatment on voltage-dependent activation and inactivation, whereas inhibitors of protein kinase C failed to alter these parameters. The combination of biochemical and electrophysiological evidence suggests that the biophysical characteristics of Kv3.1 that are important to its role in MNTB neurons, allowing them to follow high-frequency stimuli with fidelity, are largely determined by phosphorylation of the channel.

Cloning and localization of the hyperpolarization-activated cyclic nucleotide-gated channel family in rat brain.

Rhythmic firing in brain and heart is mediated by pacemaker channels that are activated by hyperpolarization and regulated directly by cyclic nucleotides. Recent work has identified a new gene family that encodes such channels, which are termed hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. In this study, we report the molecular cloning and localization by in situ hybridization of HCN1-4 in adult rat brain. The rat HCN1-4 clones show high homology to the deduced amino acid sequence of the mouse channels (>97% identity). The mRNA expression of the four channels in adult brain was evaluated in a systematic manner from the olfactory bulb to lower brain stem nuclei. Each mRNA demonstrated a unique pattern of distribution. HCN1 expression is highly enriched in cerebral cortex, hippocampus, cerebellum, and facial motor nucleus; HCN2 is highly abundant in mamillary bodies, pontine nucleus, ventral cochlear nucleus, and nucleus of the trapezoid body; HCN3 expression is most pronounced in supraoptic nucleus of hypothalamus; and HCN4 expression is most abundant in medial habenula and anterior and principal relay nuclei of the thalamus. These variations in regional specificity of HCN channels could generate important differences in neuronal pacemaker activity across brain systems.

Modification of delayed rectifier potassium currents by the Kv9.1 potassium channel subunit.

Within auditory pathways, the intrinsic electrical properties of neurons, and in particular their complement of potassium channels, play a key role in shaping the timing and pattern of action potentials produced by sound stimuli. The Kv9.1 gene encodes a potassium channel alpha subunit that is expressed in a variety of neurons, including those of the inferior colliculus. When cRNA encoding this subunit is injected into Xenopus oocytes, no functional channels are expressed. When, however, Kv9.1 is co-expressed with certain other alpha potassium channel subunits, it changes the characteristics of the currents produced by these functional channel proteins. We have found that Kv9.1 isolated from a rat brain cDNA library alters the kinetics and the voltage-dependence of activation and inactivation of Kv2.1, a channel subunit that generates slowly inactivating delayed rectifier potassium currents. The rate of activation of Kv2.1 is slowed by co-expression with Kv9.1. With Kv2.1 alone, the amplitude of evoked currents increases monotonically with increasing command potentials. In contrast, when Kv2.1 is co-expressed with Kv9.1, the amplitude of currents increases with increasing depolarization up to potentials of only approximately +60 mV, after which increasing depolarization results in a decrease in current amplitude. Currents produced by Kv2. 1 alone and by Kv2.1/Kv9.1 are both sensitive to the potassium channel blocker tetraethyl ammonium ions (TEA), but higher concentrations of TEA (20 mM) eliminate the biphasic voltage-dependence of the Kv2.1/Kv9.1 currents. Co-expression with Kv9.1 also produces an apparent negative shift in the voltage-dependence of inactivation and activation. Computer simulations of model neurons suggest that co-expression of Kv9.1 with Kv2.1 may have different effects in neurons depending on whether their firing pattern is limited by the inactivation of inward currents. In excitable cells in which the inward currents do not inactivate, co-expression with Kv9.1 could produce an inhibition of firing during sustained depolarization. In contrast, in model neurons with rapidly inactivating inward current, the change in the voltage-dependence of activation produced by Kv9.1 may allow the cells to follow high frequency stimulation more effectively.

Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Activation of a Ca2+-permeable cation channel produces a prolonged attenuation of intracellular Ca2+ release in Aplysia bag cell neurones.

1. Brief synaptic stimulation, or exposure to Conus textile venom (CtVm), triggers an afterdischarge in the bag cell neurones of Aplysia. This is associated with an elevation of intracellular calcium ([Ca2+]i) through Ca2+ release from intracellular stores and Ca2+ entry through voltage-gated Ca2+ channels and a non-selective cation channel. The afterdischarge is followed by a prolonged (approximately 18 h) refractory period during which the ability of both electrical stimulation and CtVm to trigger afterdischarges or elevate [Ca2+]i is severely attenuated. By measuring the response of isolated cells to CtVm, we have now tested the contribution of different sources of Ca2+ elevation to the onset of the prolonged refractory period. CtVm induced an increase in [Ca2+]i in both normal and Ca2+-free saline, in part by liberating Ca2+ from a store sensitive to thapsigargin or cyclopiazonic acid, but not sensitive to heparin. 3. In the presence of extracellular Ca2+, the neurones became refractory to CtVm after a single application but recovered following approximately 24 h, when CtVm could again elevate [Ca2+]i. However, this refractoriness did not develop if CtVm was applied in Ca2+-free saline. Thus, elevation of [Ca2+]i alone does not induce refractoriness to CtVm-induced [Ca2+]i elevation, but Ca2+ influx triggers this refractory-like state. 4. CtVm produces a depolarization of isolated bag cell neurones. To determine if Ca2+ influx through voltage-gated Ca2+ channels, activated during this depolarization, caused refractoriness to CtVm-induced [Ca2+]i elevation, cells were depolarized with high external potassium (60 mM), which produced a large increase in [Ca2+]i. Nevertheless, subsequent exposure of the cells to CtVm produced a normal response, suggesting that Ca2+ influx through voltage-gated Ca2+ channels does not induce refractoriness. 5. As a second test for the role of voltage-gated Ca2+ channels, these channels were blocked with nifedipine. This drug failed to prevent the onset of refractoriness to CtVm-induced [Ca2+]i elevation, providing further evidence that Ca2+ entry through voltage-gated Ca2+ channels does not initiate refractoriness. 6. To examine if Ca2+ entry through the CtVm-activated, non-selective cation channel caused refractoriness, neurones were treated with a high concentration of TTX, which blocks the cation channel. TTX protected the neurones from the refractoriness to [Ca2+]i elevation produced by CtVm in Ca2+-containing medium. 7. Using clusters of bag cell neurones in intact abdominal ganglia, we compared the ability of nifedipine and TTX to protect the cells from refractoriness to electrical stimulation. Normal, long-lasting afterdischarges could be triggered by stimulation of an afferent input after a period of exposure to CtVm in the presence of TTX. In contrast, exposure to CtVm in the presence of nifedipine resulted in refractoriness. 8. Our data indicate that Ca2+ influx through the non-selective cation channel renders cultured bag cell neurones refractory to repeated stimulation with CtVm. Moreover, the refractory period of the afterdischarge itself may also be initiated by Ca2+ entry through this cation channel.

Shaw-like potassium currents in the auditory rhombencephalon throughout embryogenesis.

The Shaw subfamily of potassium channel genes, including Kv3.1, are highly expressed within the auditory nuclei of the brainstem, where they have been implicated in the characteristic response properties of particular types of neurons. Potassium currents carried by Kv3.1 are voltage-dependent, have a high activation threshold, are slow to inactivate, and are very sensitive to 4-aminopyridine (4-AP) and tetraethylammonium (TEA). We have investigated the developmental appearance of potassium currents in cell cultures from nucleus magnocellularis and its precursor neuroblasts from the acoustico-vestibular anlage of the chicken. Whole-cell patch recordings revealed that high-threshold, sustained, outward currents were present in 91% of neuroblasts. These currents displayed high sensitivities to TEA and 4-AP. The remaining 9% of neuroblasts exhibited only transient outward currents. Most cells (74%) had both a sustained and an initial transient component of outward current. These current types were observed throughout embryogenesis, beginning with the earliest ages (embryonic day [E]2). During proliferation and migration, and early neuronal differentiation, current levels were low; they incremented gradually during the time when the first synapses occur on dendrites and increased 2- to 3-fold just before hatching, when axosomatic synapses form. These findings suggest that the Shaw subfamily of channels in nucleus magnocellularis may be involved in early neuronal development, as well as in synaptic function later on.

Role in neuronal cell migration for high-threshold potassium currents in the chicken hindbrain.

We have investigated the influence of voltage-dependent, potassium conductances on the migration of embryonic neurons, using a culture preparation taken from the acoustico-vestibular anlage long before the onset of electrical excitability and synaptic function. Whole-cell patch clamp recordings from migrating neuroblasts at Hamburger-Hamilton stage 28 (E 5.5) revealed the exclusive expression of voltage-dependent, high-threshold, outward currents, activating at potentials positive to -20 mV. These currents were completely suppressed by the potassium channel blockers, 1.0 mM tetraethylammonium chloride (TEA) or 1.0 mM 4-aminopyridine (4-AP). In control media, the active migration of individual neuroblasts was recorded at 27 +/- 6 microm per hr. Within minutes after adding either drug to the culture, normal migration completely stopped for several hours. Calcium channel blockers, omega-conotoxin (3 microM) or cadmium chloride (100 microM), slowed, but did not halt, migration, while nickel chloride (100 microM) or N-methyl-D-glucamine (1 mM) had no effect. However, within 8 hr after TEA exposure, migratory activity usually returned. This recovery was associated with the appearance of a previously undetected, low-threshold and 4-AP- sensitive potassium conductance. We suggest that high-threshold, TEA/4-AP-sensitive potassium channels may normally support the migration of these neurons, while their chronic blockade can be compensated by the appearance of novel potassium channels. Potassium currents may act in concert with N-type calcium channels to regulate neuronal migration.

Prolonged activation of mitochondrial conductances during synaptic transmission.

Although ion channels have been detected in mitochondria, scientists have not been able to record ion transport in mitochondria of intact cells. A variation of the patch clamp technique was used to record ion channel activity from intracellular organelles in the presynaptic terminal of the squid. Electron microscopy indicated that mitochondria are numerous in this terminal and are the only organelles compatible with the tips of the pipettes. Before synaptic stimulation, channel activity was infrequent and its conductance was small, although large conductances ( approximately 0.5 to 2.5 nanosiemens) could be detected occasionally. During a train of action potentials, the conductance of the mitochondrial membrane increased up to 60-fold. The conductance increased after a delay of several hundred milliseconds and continued to increase after stimulation had stopped. Recovery occurred over tens of seconds.

Cell type-specific expression of the Kv3.1 gene is mediated by a negative element in the 5' untranslated region of the Kv3.1 promoter.

The Kv3.1 potassium channel gene is restrictively expressed in the CNS, and its expression level is especially high in neurons that are able to follow synaptic inputs at high frequencies. To understand the transcriptional mechanisms controlling Kv3.1 expression, we have conducted a functional analysis of the Kv3.1 promoter in various cell lines of different tissue origins and in transgenic mice. Our results suggest that an upstream regulatory fragment coupled with the 5' untranslated region (UTR) is able to confer tissue-specific expression in both cell lines and in transgenic mice. Deletion analysis of the regulatory region carried out in cell lines reveals that a strong negatively acting element, uniquely residing in the 5' UTR (+350 to +158), appears able to confer cell type specificity on both the Kv3.1 promoter and the thymidine kinase promoter in transient transfection assays. A weak cell type-specific enhancer in the proximal region of the promoter (-123 to -71) also contributes to cell type-specific expression of the Kv3.1 gene.

hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. Roles in proliferation and volume regulation.

Human T lymphocytes express a Ca2+-activated K+ current (IK), whose roles and regulation are poorly understood. We amplified hSK4 cDNA from human T lymphoblasts, and we showed that its biophysical and pharmacological properties when stably expressed in Chinese hamster ovary cells were essentially identical to the native IK current. In activated lymphoblasts, hSK4 mRNA increased 14.6-fold (Kv1.3 mRNA increased 1.3-fold), with functional consequences. Proliferation was inhibited when Kv1.3 and IK were blocked in naive T cells, but IK block alone inhibited re-stimulated lymphoblasts. IK and Kv1.3 were involved in volume regulation, but IK was more important, particularly in lymphoblasts. hSK4 lacks known Ca2+-binding sites; however, we mapped a Ca2+-dependent calmodulin (CaM)-binding site to the proximal C terminus (Ct1) of hSK4. Full-length hSK4 produced a highly negative membrane potential (Vm) in Chinese hamster ovary cells, whereas the channels did not function when either Ct1 or the distal C terminus was deleted (Vm approximately 0 mV). Native IK (but not expressed hSK4) current was inhibited by CaM and CaM kinase antagonists at physiological Vm values, suggesting modulation by an accessory molecule in native cells. Our results provide evidence for increased roles for IK/hSK4 in activated T cell functions; thus hSK4 may be a promising therapeutic target for disorders involving the secondary immune response.