Synapses in the central nervous system undergo various short- and long-term changes in their strength, but it is often difficult to distinguish whether presynaptic or postsynaptic mechanisms are responsible for these changes. Using patch-clamp recording from giant synapses in the mouse auditory brainstem, we show here that short-term synaptic depression can be largely attributed to rapid depletion of a readily releasable pool of vesicles. Replenishment of this pool is highly dependent on the recent history of synaptic activity. High-frequency stimulation of presynaptic terminals significantly enhances the rate of replenishment. Broadening the presynaptic action potential with the potassium-channel blocker tetraethylammonium, which increases Ca2+ entry, further enhances the rate of replenishment. As this increase can be suppressed by the Ca2+-channel blocker Cd2+ or by the Ca2+ buffer EGTA, we conclude that Ca2+ influx through voltage-gated Ca2+ channels is the key signal that dynamically regulates the refilling of the releasable pool of synaptic vesicles in response to different patterns of inputs.

Malignant migrating partial seizures of infancy (MMPSI) is a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with developmental delay1. We performed exome sequencing in 3 probands with MMPSI and identified de novo gain-of-function mutations in the C-terminal domain of the KCNT1 potassium channel. We sequenced KCNT1 in 9 additional patients with MMPSI and identified mutations in 4 of them, in total identifying mutations in 6 out of 12 unrelated patients. Functional studies showed that the mutations led to constitutive activation of the channel, mimicking the effects of phosphorylation of the C-terminal domain by protein kinase C. In addition to regulating ion flux, KCNT1 has a non conducting function as its C terminus interacts with cytoplasmic proteins involved in developmental signaling pathways. These results provide a target for future diagnostic approaches and research in this devastating condition.

Using a combination of patch‐clamp, in situ hybridization and computer simulation techniques, we have analysed the contribution of potassium channels to the ability of a subset of mouse auditory neurones to fire at high frequencies. Voltage‐clamp recordings from the principal neurones of the medial nucleus of the trapezoid body (MNTB) revealed a low‐threshold dendrotoxin (DTX)‐sensitive current (ILT) and a high‐threshold DTX‐insensitive current (IHT). I HT displayed rapid activation and deactivation kinetics, and was selectively blocked by a low concentration of tetraethylammonium (TEA; 1 mm). The physiological and pharmacological properties of IHT very closely matched those of the Shaw family potassium channel Kv3.1 stably expressed in a CHO cell line. An mRNA probe corresponding to the C‐terminus of the Kv3.1 channel strongly labelled MNTB neurones, suggesting that this channel is expressed in these neurones. TEA did not alter the ability of MNTB neurones to follow stimulation up to 200 Hz, but specifically reduced their ability to follow higher frequency impulses. A computer simulation, using a model cell in which an outward current with the kinetics and voltage dependence of the Kv3.1 channel was incorporated, also confirmed that the Kv3.1‐ like current is essential for cells to respond to a sustained train of high‐frequency stimuli. We conclude that in mouse MNTB neurones the Kv3.1 channel contributes to the ability of these cells to lock their firing to high‐frequency inputs.

Neuronal stressors such as hypoxia and firing of action potentials at very high frequencies cause intracellular Na+ to rise and ATP to be consumed faster than it can be regenerated. We report the cloning of a gene encoding a K+ channel, Slick, and demonstrate that functionally it is a hybrid between two classes of K+ channels, Na+-activated (KNa) and ATP-sensitive (KATP) K+ channels. The Slick channel is activated by intracellular Na+ and Cl- and is inhibited by intracellular ATP. Slick is widely expressed in the CNS and is detected in heart. We identify a consensus ATP binding site near the C terminus of the channel that is required for ATP and its nonhydrolyzable analogs to reduce open probability. The convergence of Na+, Cl-, and ATP sensitivity in one channel may endow Slick with the ability to integrate multiple indicators of the metabolic state of a cell and to adjust electrical activity appropriately.

Recent evidence suggests that intracellular Zn 2؉ accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn 2؉ that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn 2؉ that are mobilized in response to stimulation. One such Zn 2؉ pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn 2؉ release from one resulting in apparent net Zn 2؉ transfer to the other. Further studies found evidence for complex and potent effects of Zn 2؉ on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of ⌬ m), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O2 consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O2 consumption and increased ROS generation. Finally, strong mobilization of proteinbound Zn 2؉ appeared to induce partial loss of ⌬ m, suggesting that movement of Zn 2؉ between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

Potassium channels catalyse the permeation of K+ ions across cellular membranes and are identified by a common structural motif, a highly conserved signature sequence of eight amino acids in the P domain of each channel's pore-forming alpha-subunit. Here we describe a novel K+ channel (TOK1) from Saccharomyces cerevisiae that contains two P domains within one continuous polypeptide. Xenopus laevis oocytes expressing the channel exhibit a unique, outwardly rectifying, K(+)-selective current. The channel is permeable to outward flow of ions at membrane potentials above the K+ equilibrium potential; its conduction-voltage relationship is thus sensitive to extracellular K+ ion concentration. In excised membrane patches, external divalent cations block the channel in a voltage-dependent manner, and their removal in this configuration allows inward channel current. These attributes are similar to those described for inwardly rectifying K+ channels, but in the opposite direction, a previously unrecognized channel behaviour. Our results identify a new class of K+ channel which is distinctive in both its primary structure and functional properties. Structural homologues of the channel are present in the genome of Caenorhabditis elegans.

The gene for hSK4, a novel human small conductance calcium-activated potassium channel, or SK channel, has been identified and expressed in Chinese hamster ovary cells. In physiological saline hSK4 generates a conductance of approximately 12 pS, a value in close agreement with that of other cloned SK channels. Like other members of this family, the polypeptide encoded by hSK4 contains a previously unnoted leucine zipper-like domain in its C terminus of unknown function. hSK4 appears unique, however, in its very high affinity for Ca 2؉ (EC 50 of 95 nM) and its predominant expression in nonexcitable tissues of adult animals. Together with the relatively low homology of hSK4 to other SK channel polypeptides (approximately 40% identical), these data suggest that hSK4 belongs to a novel subfamily of SK channels.In mammals, small conductance calcium-activated potassium channels, or SK channels, are thought to underlie currents that have been described in a wide range of tissues, including brain (1-13), peripheral nervous system (14-16), skeletal muscle (17-19), adrenal chromaffin cells (20)(21)(22), leukocytes (23-28), erythrocytes (29-32), colon (33, 34), and airway epithelia (35,36). Pharmacologically, certain types of SK channels have been distinguished by their sensitivities to the bee venom apamin (5, 7-23, 37), whereas other functionally related conductances appear insensitive (7,24,27,34). Features that distinguish members of this family from their closest phenotypic neighbors, the maxi-K calcium-activated, or BK, potassium channels, are the SK channels' low conductance (less than 50 pS), the weak or negligible dependence of their activity on membrane voltage, and their high affinity for Ca 2ϩ (EC 50 Ͻ 1 M) (3, 19-23, 25, 26, 33-40).Fragments of SK genes first were identified in computerbased searches of GenBank's database of expressed sequence tags (ESTs) for cDNAs encoding sequences resembling the pore domains of known families of K ϩ channels (41). We have extended this work by identifying ESTs including the gene encoding human SK4 (hSK4), a member of a novel subfamily of SK channels, and expressing one of these cDNAs in Chinese hamster ovary cells. In addition, we cloned the full-length gene of rSK1 (41).Members of the first subfamily to be described are predominantly expressed in excitable tissues and are half-maximally activated at cytosolic free Ca 2ϩ concentrations in the range of 600-700 nM (41). The hSK4 channel differs from these in that its transcript is found in nonexcitable tissues and is halfactivated at 95 nM free Ca 2ϩ , indicating it is likely to be open at resting levels of Ca 2ϩ in certain types of cells. The hyperpolarization resulting from the activity of hSK4 suggests that this channel could regulate electrogenic transport. METHODSCloning of SK Genes. The two P regions of the yeast TOK channel were used to screen the EST database of GenBank using the BLAST algorithm (42). One of the ESTs that was identified as a novel potential mammalian K ϩ channel cDNA was labeled by random prim...

Summary The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells and their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.