Hiroaki Misonou

Regulation of ion channel localization and phosphorylation by neuronal activity

Voltage-dependent Kv2.1 K+ channels, which mediate delayed rectifier Kv currents (IK), are expressed in large clusters on the somata and dendrites of principal pyramidal neurons, where they regulate neuronal excitability. Here we report activity-dependent changes in the localization and biophysical properties of Kv2.1. In the kainate model of continuous seizures in rat, we find a loss of Kv2.1 clustering in pyramidal neurons in vivo. Biochemical analysis of Kv2.1 in the brains of these rats shows a marked dephosphorylation of Kv2.1. In cultured rat hippocampal pyramidal neurons, glutamate stimulation rapidly causes dephosphorylation of Kv2.1, translocation of Kv2.1 from clusters to a more uniform localization, and a shift in the voltage-dependent activation of IK. An influx of Ca2+ leading to calcineurin activation is both necessary and sufficient for these effects. Our finding that neuronal activity modifies the phosphorylation state, localization and function of Kv2.1 suggests an important link between excitatory neurotransmission and the intrinsic excitability of pyramidal neurons.

Distinct intramembrane cleavage of the beta-amyloid precursor protein family resembling gamma-secretase-like cleavage of Notch

The intramembrane cleavage of β-amyloid precursor protein by γ-secretase is the final step in the generation of amyloid β-protein. A 59- or 57-residue C-terminal fragment called CTFγ is produced concomitantly. Putative CTFγ generated in rat brain membrane preparations was purified and sequenced. Instead of CTFγ, shorter 50- and 49-residue fragments were identified. In addition, we found similar C-terminal fragments of β-amyloid precursor-like proteins 1 and 2; these were also cleaved at corresponding sites. This newly identified cleavage occurs at a site two to five residues inside the cytoplasmic membrane boundary, which is very similar to γ-secretase-like cleavage of Notch 1.

Calcium- and Metabolic State-Dependent Modulation of the Voltage-Dependent Kv2.1 Channel Regulates Neuronal Excitability in Response to Ischemia

Ischemic stroke is often accompanied by neuronal hyperexcitability (i.e., seizures), which aggravates brain damage. Therefore, suppressing stroke-induced hyperexcitability and associated excitoxicity is a major focus of treatment for ischemic insults. Both ATP-dependent and Ca2+-activated K+ channels have been implicated in protective mechanisms to suppress ischemia-induced hyperexcitability. Here we provide evidence that the localization and function of Kv2.1, the major somatodendritic delayed rectifier voltage-dependent K+ channel in central neurons, is regulated by hypoxia/ischemia-induced changes in metabolic state and intracellular Ca2+ levels. Hypoxia/ischemia in rat brain induced a dramatic dephosphorylation of Kv2.1 and the translocation of surface Kv2.1 from clusters to a uniform localization. In cultured rat hippocampal neurons, chemical ischemia (CI) elicited a similar dephosphorylation and translocation of Kv2.1. These events were reversible and were mediated by Ca2+ release from intracellular stores and calcineurin-mediated Kv2.1 dephosphorylation. CI also induced a hyperpolarizing shift in the voltage-dependent activation of neuronal delayed rectifier currents (IK), leading to enhanced IK and suppressed neuronal excitability. The IK blocker tetraethylammonium reversed the ischemia-induced suppression of excitability and aggravated ischemic neuronal damage. Our results show that Kv2.1 can act as a novel Ca2+- and metabolic state-sensitive K+ channel and suggest that dynamic modulation of IK/Kv2.1 in response to hypoxia/ischemia suppresses neuronal excitability and could confer neuroprotection in response to brief ischemic insults.

Immunolocalization of the Ca2+-Activated K+ Channel Slo1 in Axons and Nerve Terminals of Mammalian Brain and Cultured Neurons

Ca2+‐activated voltage‐dependent K+ channels (Slo1, KCa1.1, Maxi‐K, or BK channel) play a crucial role in controlling neuronal signaling by coupling channel activity to both membrane depolarization and intracellular Ca2+ signaling. In mammalian brain, immunolabeling experiments have shown staining for Slo1 channels predominantly localized to axons and presynaptic terminals of neurons. We have developed anti‐Slo1 mouse monoclonal antibodies that have been extensively characterized for specificity of staining against recombinant Slo1 in heterologous cells, and native Slo1 in mammalian brain, and definitively by the lack of detectable immunoreactivity against brain samples from Slo1 knockout mice. Here we provide precise immunolocalization of Slo1 in rat brain with one of these monoclonal antibodies and show that Slo1 is accumulated in axons and synaptic terminal zones associated with glutamatergic synapses in hippocampus and GABAergic synapses in cerebellum. By using cultured hippocampal pyramidal neurons as a model system, we show that heterologously expressed Slo1 is initially targeted to the axonal surface membrane, and with further development in culture, become localized in presynaptic terminals. These studies provide new insights into the polarized localization of Slo1 channels in mammalian central neurons and provide further evidence for a key role in regulating neurotransmitter release in glutamatergic and GABAergic terminals. J. Comp. Neurol. 496:289–302, 2006.

Determinants of Voltage-Gated Potassium Channel Surface Expression and Localization in Mammalian Neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K+ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca2+ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.

Requirement of subunit co-assembly and ankyrin-G for M-channel localization at the axon initial segment

The potassium channel subunits KCNQ2 and KCNQ3 are believed to underlie the M current of hippocampal neurons. The M-type potassium current plays a key role in the regulation of neuronal excitability; however, the subcellular location of the ion channels underlying this regulation has been controversial. We report here that KCNQ2 and KCNQ3 subunits are localized to the axon initial segment of pyramidal neurons of adult rat hippocampus and in cultured hippocampal neurons. We demonstrate that the localization of the KCNQ2/3 channel complex to the axon initial segment is favored by co-expression of the two channel subunits. Deletion of the ankyrin-G-binding motif in both the KCNQ2 and KCNQ3 C-terminals leads to the disappearance of the complex from the axon initial segment, albeit the channel complex remains functional and still reaches the plasma membrane. We further show that although heteromeric assembly of the channel complex favours localization to the axon initial segment, deletion of the ankyrin-G-binding motif in KCNQ2 alone does not alter the subcellular localization of KCNQ2/3 heteromers. By contrast, deletion of the ankyrin-G-binding motif in KCNQ3 significantly reduces AIS enrichment of the complex, implicating KCNQ3 as a major determinant of M channel localization to the AIS.

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation

Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.

Regulation of intrinsic excitability in hippocampal neurons by activity-dependent modulation of the Kv2.1 potassium channel

KV2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (Kv) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of KV2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the KV2.1-component of delayed rectifier K+ (IK) currents. In computer models of hippocampal neurons, these glutamate-stimulated shifts in the gating of the KV2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate-stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of KV1.1-containing channels. These studies together demonstrate a specific contribution of modulation of KV2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.

Dynamic regulation of the Kv2.1 voltage-gated potassium channel during brain ischemia through neuroglial interaction.

The physiological significance of neuroglial interactions in the CNS has been emphasized in neurological conditions such as epilepsy and brain ischemia. The Kv2.1 voltage-gated potassium channel is unique in its ability to form large clusters in the plasma membrane of neuronal cell bodies. We have previously shown that brain ischemia causes rapid dephosphorylation of Kv2.1 subunits and resultant activation of the ion channel function. However, the physiological significance of the channel clustering is unknown. Here we present evidence that clustered Kv2.1 channels in the neuronal plasma membrane are juxtaposed to axosomatic synapses and associated with astrocytic processes expressing high levels of glutamate transporters. In acute cortical slices, ischemic stress rapidly resulted in the dephosphorylation and dispersion of Kv2.1. Selective inhibition of metabolism in astrocytes was sufficient to induce Kv2.1 dephosphorylation in neurons. Interestingly, these effects were blocked by the antagonists of ionotropic glutamate receptors, indicating the involvement of glutamate as the signal mediator between astrocytes and neurons. Furthermore, the pharmacological inhibition of glial glutamate transporter GLT-1 induced the similar Kv2.1 dephosphorylation, whereas exogenous glutamate alone was not efficacious. These results suggest that ischemic stress rapidly causes the dysfunction of glutamate transporters in astrocytes and resultant accumulation of glutamate in the extracellular space. The elevated glutamate may subsequently activate ionotropic glutamate receptors and result in the dephosphorylation of Kv2.1 in neurons. These findings implicate that Kv2.1 clusters are strategically situated at neuroglial junctions to achieve the rapid modulation after ischemic stress via glutamate signaling.

Homeostatic Regulation of Neuronal Excitability by K+ Channels in Normal and Diseased Brains

K+-selective ion channels are critical determinants of membrane excitability in neuronal cells. Like many other cells in our body, neuronal cells have a propensity to maintain their homeostasis. Action potential firing is the most important function to maintain in brain neurons, as they are the elements of neural networks. If one element fires action potentials at an abnormally high rate, the entire network could become epileptic. Therefore, brain neurons adjust their intrinsic membrane excitability to maintain the firing rate within their own optimal operational range. When a neuron receives an enormous input, it will reduce the membrane excitability to prevent overshooting. When it is deprived of stimulus, the membrane becomes more excitable to avoid total quiescence. The homeostatic regulation of intrinsic excitability provides stability to the neural network in the face of dynamic and plastic synaptic inputs. In the past decade, we have learned that neurons achieve this type of homeostatic regulation through a variety of ion channels, including K+ channels. It has also become clear that under certain pathological conditions, these homeostatic mechanisms provide neuroprotection. In this article, I will review recent advances in our understanding of K+ channel—mediated homeostatic regulation of neuronal excitability and discuss involvement of these channels in hyperexcitable diseases where they provide neuroprotection.