regulates the Kv4.2 potassium channel by direct phosphorylation of the pore-forming subunit. Am J Physiol Cell Physiol 290: C852-C861, 2006. First published October 26, 2005 doi:10.1152 doi:10. /ajpcell.00358.2005.2 is the primary pore-forming subunit encoding A-type currents in many neurons throughout the nervous system, and it also contributes to the transient outward currents of cardiac myocytes. A-type currents in the dendrites of hippocampal CA1 pyramidal neurons are regulated by activation of ERK/MAPK, and Kv4.2 is the likely pore-forming subunit of that current. We showed previously that Kv4.2 is directly phosphorylated at three sites by ERK/MAPK (T602, T607, and S616). In this study we determined whether direct phosphorylation of Kv4.2 by ERK/ MAPK is responsible for the regulation of the A-type current observed in neurons. We made site-directed mutants, changing the phosphosite serine (S) or threonine (T) to aspartate (D) to mimic phosphorylation. We found that the T607D mutation mimicked the electrophysiological changes elicited by ERK/MAPK activation in neurons: a rightward shift of the activation curve and an overall reduction in current compared with wild type (WT). Surprisingly, the S616D mutation caused the opposite effect, a leftward shift in the activation voltage. K ϩ channel-interacting protein (KChIP)3 ancillary subunit coexpression with Kv4.2 was necessary for the T607D effect, as the T607D mutant when expressed in the absence of KChIP3 was not different from WT Kv4.2. These data suggest that direct phosphorylation of Kv4.2 at T607 is involved in the dynamic regulation of the channel function by ERK/MAPK and an interaction of the primary subunit with KChIP is also necessary for this effect. Overall these studies provide new insights into the structure-function relationships for MAPK regulation of membrane ion channels. K ϩ channel-interacting protein; kinase; neurons; A-type current MANY STUDIES HAVE SHOWN THAT ERK is important for regulation of neuronal function, particularly playing a role in the regulation of synaptic plasticity and long-term memory formation (3,13,14,30,46,48). Considerable evidence is accumulating that ERK activation plays a role in the regulation of postsynaptic excitability, specifically operating in the context of synaptic plasticity (40,46,48). One potential mechanism of this regulation by ERK is indirect, by long-term modulation of cell properties through the control of gene transcription and regulation of channel gene expression (9). Another possible mechanism by which ERK might modulate neuronal excitability is through direct regulation of membrane ion channels that regulate the membrane potential and thereby intrinsic membrane properties.In our recent studies, we have focused on regulation of ion channels by ERK because modulation of excitability may be a critical factor that ultimately controls the induction of longlasting changes in synaptic strength. One possible direct target of ERK is the K ϩ channel Kv4.2, which encodes a transient A-type K ϩ current that...

Adult murine bone marrow hematopoietic stem cells (HSCs) can be purified by sorting Hoechst 33342-extruding side population (SP) cells. Herein we investigated whether SP cells reside within embryonic tissues and exhibit hematopoietic progenitor activity. We isolated yolk sac (YS) and embryonic tissues 7.5 to 11.5 days after coitus (dpc), resolved an SP in each, and demonstrated that these SP cells exhibit distinct phenotypic and functional characteristics throughout development. YS and embryonic SP isolated 8.0 dpc expressed vascular endothelial-cadherin (VE-cadherin) and vascular endothelial receptor 2 (Flk-1), markers not expressed by bone marrow SP but expressed by endothelial cells and progenitors. SP at this stage did not express CD45 or produce hematopoietic colonies in vitro. In contrast, SP isolated 9.5 to 11.5 dpc contained a significantly higher proportion of cells expressing cKit and CD45, markers highly expressed by bone marrow SP. Furthermore, YS SP isolated 9.5 to 11.5 dpc demonstrated 40-to 90-fold enrichment for hematopoietic progenitor activity over unfractionated tissue. Our data indicate that YS and embryonic SP cells detected prior to the onset of circulation express the highest levels of endothelial markers and do not generate blood cells in vitro; however, as development progresses, they acquire hematopoietic potential and phenotypic characteristics similar to those of bone marrow SP.

In cerebellar granule (CG) cells and many other neurons, A-type potassium currents play an important role in regulating neuronal excitability, firing patterns, and activity-dependent plasticity. Protein biochemistry has identified dipeptidyl peptidase-like protein 6 (DPP6) as an auxiliary subunit of Kv4-based A-type channels and thus a potentially important regulator of neuronal excitability. In this study, we used an RNA interference (RNAi) strategy to examine the role DPP6 plays in forming and shaping the electrophysiological properties of CG cells. DPP6 RNAi delivered by lentiviral vectors effectively disrupts DPP6 protein expression in CG cells. In response to the loss of DPP6, I SA peak conductance amplitude is reduced by Ͼ85% in parallel with a dramatic reduction in the level of I SA channel protein complex found in CG cells. The I SA channels remaining in CG cells after suppression of DPP6 show alterations in gating similar to Kv4 channels expressed in heterologous systems without DPP6. In addition to these effects on A-type current, we find that loss of DPP6 has additional effects on input resistance and Na ϩ channel conductance that combine with the effects on I SA to produce a global change in excitability. Overall, DPP6 expression seems to be critical for the expression of a high-frequency electrophysiological phenotype in CG cells by increasing leak conductance, A-type current levels and kinetics, and Na ϩ current amplitude.

The metabotropic glutamate receptors (mGluRs) have been predicted to have a classical seven transmembrane domain structure similar to that seen for members of the G-protein-coupled receptor (GPCR) superfamily. However, the mGluRs (and other members of the family C GPCRs) show no sequence homology to the rhodopsinlike GPCRs, for which this seven transmembrane domain structure has been experimentally confirmed. Furthermore, several transmembrane domain prediction algorithms suggest that the mGluRs have a topology that is distinct from these receptors. In the present study, we set out to test whether mGluR5 has seven true transmembrane domains. Using a variety of approaches in both prokaryotic and eukaryotic systems, our data provide stong support for the proposed seven transmembrane domain model of mGluR5. We propose that this membrane topology can be extended to all members of the family C GPCRs.Glutamate is the primary excitatory neurotransmitter of the central nervous system and activates ligand-gated ion channels or ionotropic glutamate receptors (iGluRs) and G-protein-coupled metabotropic glutamate receptors (mGluRs). 1The mGluRs modulate synaptic transmission and neuronal excitability throughout the peripheral and central nervous system. They have been classified into three groups based on sequence homology, pharmacology, and signal transduction coupling. Group I mGluRs, consisting of mGluR1 and mGluR5, uniformly couple to phospholipase C, while group II and III mGluRs, composed of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8, inhibit adenylyl cyclase activity in heterologous expression systems (1, 2).Since their cloning, the structure-function relationships of mGluRs have garnered extensive interest. Relatively recent classification of G-protein-coupled receptors (GPCRs) has categorized mGluRs as family C GPCRs characterized structurally by a large N-terminal extracellular domain and consisting of mGluRs, GABA B receptors, Ca 2ϩ -sensing receptors, and certain pheromone receptors. The large N-terminal extracellular domain of mGluRs exhibits distant homology to bacterial amino acid periplasmic binding proteins, suggesting the domain is responsible for ligand binding (3). Structural modeling of mGluRs based on this homology accurately predicts important residues for ligand binding (4). Exchange of this domain among mGluRs also endows specific ligand binding properties (5-7). Recently, crystal structure determination of the N-terminal domain with bound ligand has definitively confirmed its role in ligand binding and conformational changes of the domain with binding have made several predictions for potential mechanisms for receptor activation (8).While the structure of the ligand binding N-terminal domain has been clearly resolved, little data exists on the structure and membrane topology of the subsequent transmembrane domains. The complete structure of the canonical GPCR, rhodopsin, has recently been solved and confirms a long line of studies delineating seven membrane spanning ␣-helices (9 -12). Based...

The somatodendritic A-current, I SA , in hippocampal CA1 pyramidal neurons regulates the processing of synaptic inputs and the amplitude of back propagating action potentials into the dendritic tree, as well as the action potential firing properties at the soma. In this study, we have used RNA interference and over-expression to show that expression of the Kv4.2 gene specifically regulates the I SA component of A-current in these neurons. In dissociated hippocampal pyramidal neuron cultures, or organotypic cultured CA1 pyramidal neurons, the expression level of Kv4.2 is such that the I SA channels are maintained in the population at a peak conductance of approximately 950 pS/pF. Suppression of Kv4.2 transcripts in hippocampal pyramidal neurons using an RNA interference vector suppresses I SA current by 60% in 2 days, similar to the effect of expressing dominant-negative Kv4 channel constructs. Increasing the expression of Kv4.2 in these neurons increases the level of I SA to 170% of the normal set point without altering the biophysical properties. Our results establish a specific role for native Kv4.2 transcripts in forming and maintaining I SA current at characteristic levels in hippocampal pyramidal neurons.