Localization of SK2 channels relative to excitatory synaptic sites in the mouse developing Purkinje cells.
ABSTRACT: Small-conductance, Ca(2+)-activated K(+) (SK) channels regulate neuronal excitability in a variety of ways. To understand their roles in different neuronal subtypes it is important to determine their precise subcellular distribution. Here, we used biochemical, light microscopy immunohistochemical and immunoelectron microscopy techniques, combined with quantitative approaches, to reveal the expression and subcellular localization patterns of SK2 in the developing cerebellum. Using western blots, the SK2 protein showed a progressive increase during postnatal development. At the light microscopic level, SK2 immunoreactivity was very prominent in the developing Purkinje cells (PC), particularly in the molecular layer (ML). Electron microscopy revealed that throughout development SK2 was mostly detected at the extrasynaptic and perisynaptic plasma membrane of dendritic shafts and dendritic spines of PCs. However, there was some localization at axon terminals as well. Quantitative analyses and 3D reconstructions further revealed a progressive developmental change of SK2 on the surface of PCs from dendritic shafts to dendritic spines. Together, these results indicate that SK2 channels undergo dynamic spatial regulation during cerebellar development, and this process is associated with the formation and maturation of excitatory synaptic contacts to PCs.
Project description:SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse hippocampal area CA1 pyramidal neurons and influence synaptic responses, plasticity and learning. The Sk2 gene (also known as Kcnn2) encodes two isoforms that differ only in the length of their N-terminal domains. SK2-long (SK2-L) and SK2-short (SK2-S) are coexpressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-S only mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to excitatory postsynaptic potentials was absent in SK2-S only mice and was restored by SK2-L re-expression. Blocking SK channels increased the amount of long-term potentiation induced in area CA1 in slices from wild-type mice but had no effect in slices from SK2-S only mice. Furthermore, SK2-S only mice outperformed wild-type mice in the novel object recognition task. These results indicate that SK2-L directs synaptic SK2-containing channel expression and is important for normal synaptic signaling, plasticity and learning.
Project description:Mouse CA1 pyramidal neurons express apamin-sensitive SK2-containing channels in the post-synaptic membrane, positioned close to NMDA-type (N-methyl-D-aspartate) glutamate receptors. Activated by synaptically evoked NMDAR-dependent Ca(2+) influx, the synaptic SK2-containing channels modulate excitatory post-synaptic responses and the induction of synaptic plasticity. In addition, their activity- and protein kinase A-dependent trafficking contributes to expression of long-term potentiation (LTP). We have identified a novel synaptic scaffold, MPP2 (membrane palmitoylated protein 2; p55), a member of the membrane-associated guanylate kinase (MAGUK) family that interacts with SK2-containing channels. MPP2 and SK2 co-immunopurified from mouse brain, and co-immunoprecipitated when they were co-expressed in HEK293 cells. MPP2 is highly expressed in the post-synaptic density of dendritic spines on CA1 pyramidal neurons. Knocking down MPP2 expression selectively abolished the SK2-containing channel contribution to synaptic responses and decreased LTP. Thus, MPP2 is a novel synaptic scaffold that is required for proper synaptic localization and function of SK2-containing channels.
Project description:We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development.
Project description:Long-term potentiation (LTP) of synaptic strength at Schaffer collateral synapses has largely been attributed to changes in the number and biophysical properties of AMPA receptors (AMPARs). Small-conductance Ca(2+)-activated K(+) channels (SK2 channels) are functionally coupled with NMDA receptors (NMDARs) in CA1 spines such that their activity modulates the shape of excitatory postsynaptic potentials (EPSPs) and increases the threshold for induction of LTP. Here we show that LTP induction in mouse hippocampus abolishes SK2 channel activity in the potentiated synapses. This effect is due to SK2 channel internalization from the postsynaptic density (PSD) into the spine. Blocking PKA or cell dialysis with a peptide representing the C-terminal domain of SK2 that contains three known PKA phosphorylation sites blocks the internalization of SK2 channels after LTP induction. Thus the increase in AMPARs and the decrease in SK2 channels combine to produce the increased EPSP underlying LTP.
Project description:Compartmentalization of Ca(2+) between dendritic spines and shafts is governed by diffusion barriers and a range of Ca(2+) extrusion mechanisms. The distinct contribution of different Ca(2+) clearance systems to Ca(2+) compartmentalization in dendritic spines versus shafts remains elusive. We applied a combination of ultrastructural and functional imaging methods to assess the subcellular distribution and role of NCX1 in rat CA1 pyramidal cells. Quantitative electron microscopic analysis of preembedding immunogold reactions revealed uniform densities of NCX1 along the shafts of apical and basal dendrites, but densities in dendritic shafts were approximately seven times higher than in dendritic spines. In line with these results, two-photon imaging of synaptically activated Ca(2+) transients during NCX blockade showed preferential action localized to the dendritic shafts for NCXs in regulating spine-dendrite coupling.
Project description:In mouse hippocampal CA1 pyramidal neurons, the activity of synaptic small-conductance Ca(2+)-activated K(+) channels type 2 (SK2 channels) provides a negative feedback on N-methyl-D-aspartate receptors (NMDARs), reestablishing Mg(2+) block that reduces Ca(2+) influx. The well-established role of NMDARs in ischemia-induced excitotoxicity led us to test the neuroprotective effect of modulating SK2 channel activity following cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Administration of the SK channel positive modulator, 1-ethyl-benzimidazolinone (1-EBIO), significantly reduced CA1 neuron cell death and improved CA/CPR-induced cognitive outcome. Electrophysiological recordings showed that CA/CPR-induced ischemia caused delayed and sustained reduction of synaptic SK channel activity, and immunoelectron microscopy showed that this is associated with internalization of synaptic SK2 channels, which was prevented by 1-EBIO treatment. These results suggest that increasing SK2 channel activity, or preventing ischemia-induced loss of synaptic SK2 channels, are promising and novel approaches to neuroprotection following cerebral ischemia.
Project description:Although the extracellular matrix plays an important role in regulating use-dependent synaptic plasticity, the underlying molecular mechanisms are poorly understood. Here we examined the synaptic function of hyaluronic acid (HA), a major component of the extracellular matrix. Enzymatic removal of HA with hyaluronidase reduced nifedipine-sensitive whole-cell Ca(2+) currents, decreased Ca(2+) transients mediated by L-type voltage-dependent Ca(2+) channels (L-VDCCs) in postsynaptic dendritic shafts and spines, and abolished an L-VDCC-dependent component of long-term potentiation (LTP) at the CA3-CA1 synapses in the hippocampus. Adding exogenous HA, either by bath perfusion or via local delivery near recorded synapses, completely rescued this LTP component. In a heterologous expression system, exogenous HA rapidly increased currents mediated by Ca(v)1.2, but not Ca(v)1.3, subunit-containing L-VDCCs, whereas intrahippocampal injection of hyaluronidase impaired contextual fear conditioning. Our observations unveil a previously unrecognized mechanism by which the perisynaptic extracellular matrix influences use-dependent synaptic plasticity through regulation of dendritic Ca(2+) channels.
Project description:Small conductance Ca(2+)-activated K(+) type 2 (SK2) channels are expressed in the postsynaptic density of CA1 neurons where they are activated by synaptically evoked Ca(2+) influx to limit the size of EPSPs and spine Ca(2+) transients. At Schaffer collateral synapses, the induction of long-term potentiation (LTP) increases the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)-mediated contribution to synaptic transmission and decreases the synaptic SK2 channel contribution through protein kinase A-dependent channel endocytosis. Using a combination of electrophysiology and immunoelectron microscopy in mice, the relationship between the dynamics of spine SK2 channels and AMPARs was investigated. Unlike AMPARs, synaptic SK2 channels under basal conditions do not rapidly recycle. Furthermore, SK2 channels occupy a distinct population of endosomes separate from AMPARs. However, blocking vesicular exocytosis or the delivery of synaptic GluA1-containing AMPARs during the induction of LTP blocks SK2 channel endocytosis. By approximately 2 h after the induction of LTP, synaptic SK2 channel expression and function are restored. Thus, LTP-dependent endocytosis of SK2 is coupled to LTP-dependent AMPA exocytosis, and the approximately 2 h window after the induction of LTP during which synaptic SK2 activity is absent may be important for consolidating the later phases of LTP.
Project description:Small-conductance Ca(2+)-activated K(+) channels (SK channels) modulate excitability and curtail excitatory postsynaptic potentials (EPSPs) in neuronal dendrites. Here, we demonstrate long-lasting plasticity of intrinsic excitability (IE) in dendrites that results from changes in the gain of this regulatory mechanism. Using dendritic patch-clamp recordings from rat cerebellar Purkinje cells, we find that somatic depolarization or parallel fiber (PF) burst stimulation induce long-term amplification of synaptic responses to climbing fiber (CF) or PF stimulation and enhance the amplitude of passively propagated sodium spikes. Dendritic plasticity is mimicked and occluded by the SK channel blocker apamin and is absent in Purkinje cells from SK2 null mice. Triple-patch recordings from two dendritic sites and the soma and confocal calcium imaging studies show that local stimulation limits dendritic plasticity to the activated compartment of the dendrite. This plasticity mechanism allows Purkinje cells to adjust the SK2-mediated control of dendritic excitability in an activity-dependent manner.
Project description:Gated solely by activity-induced changes in intracellular calcium, small-conductance potassium channels (SKs) are critical for a variety of functions in the CNS, from learning and memory to rhythmic activity and sleep. While there is a wealth of information on SK2 gating, kinetics, and Ca(2+) sensitivity, little is known regarding the regulation of SK2 subcellular localization. We report here that synaptic SK2 levels are regulated by the E3 ubiquitin ligase UBE3A, whose deficiency results in Angelman syndrome and overexpression in increased risk of autistic spectrum disorder. UBE3A directly ubiquitinates SK2 in the C-terminal domain, which facilitates endocytosis. In UBE3A-deficient mice, increased postsynaptic SK2 levels result in decreased NMDA receptor activation, thereby impairing hippocampal long-term synaptic plasticity. Impairments in both synaptic plasticity and fear conditioning memory in UBE3A-deficient mice are significantly ameliorated by blocking SK2. These results elucidate a mechanism by which UBE3A directly influences cognitive function.