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Biomedical subjects

Antonius M J VanDongen

Publications and source records attributed to Antonius M J VanDongen.

11 recordsLinked to original sources
PubMed2005

Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel.

K+ channels achieve exquisite ion selectivity without jeopardizing efficient permeation by employing multiple, interacting K+-binding sites. Introduction ofa cadmium (Cd2+)-binding site in the external vestibule of Kv2.1 (drk1), allowed us to functionally characterize a binding site for external monovalent cations. Permeant ions displayed higher affinity for this site than non-permeant monovalent cations, although the selectivity profile was different from that of the channel. Point mutations identified the highly conserved aspartate residue immediately following the selectivity filter as a critical determinant of the antagonism between external K+ and Cd2+ ions. A conservative mutation at this position (D378E) significantly affected the open-state stability. Moreover, the mean open time was found to be modulated by external K+ concentration, suggesting a coupling between channel closing and the permeation process. Reducing the Rb+ conductance by mutating the selectivity filter to the sequence found inKv4.1, also significantly reduced the effectiveness ofRb+ ions to antagonize Cd2+ inhibition, thereby implicating the selectivity filter as the site at which K+ions exert their antagonistic effect on Cd2+ block. The equivalent of D378 in KcsA, D80, takes part in an inter-subunit hydrogen-bond network that allows D80to functionally interact with the selectivity filter. The results suggest that external K+ ions antagonize Cd2+inhibition (in I379C) and modulate the mean open time(in the wild-type Kv2.1) by altering the occupancy profile of the K+-binding sites in the selectivity filter.

Animals↗
PubMed2005

The nonkinase phorbol ester receptor alpha 1-chimerin binds the NMDA receptor NR2A subunit and regulates dendritic spine density.

Abnormalities in dendritic spines have long been associated with cognitive dysfunction and neurodevelopmental delay, whereas rapid changes in spine shape underlie synaptic plasticity. The key regulators of cytoskeletal reorganization in dendrites and spines are the Rho GTPases, which modify actin polymerization in response to synaptic signaling. Rho GTPase activity is modulated by multiple regulatory proteins, some of which have been found to associate with proteins localized to spines. Here, we show that the nonkinase phorbol ester receptor alpha1-chimerin is present in dendrites and spines, where it binds to the NMDA receptor NR2A subunit in a phorbol ester-dependent manner. Alpha1-chimerin contains a GTPase activating (GAP) domain, with activity toward the Rho family member Rac1. Overexpression of alpha1-chimerin in cultured hippocampal neurons inhibits formation of new spines and removes existing spines. This reduction in spine density is mediated by Rac1 inhibition, because it depends critically on the presence of a functional GAP domain. Conversely, depletion of alpha1-chimerin leads to an increase in spine density, indicating that a basal inhibition of Rac1 maintains the number of spines at a submaximal level. The ability of alpha1-chimerin to modulate spine number requires an interaction with the NMDA receptor, because an alpha1-chimerin mutant that binds weakly to NR2A fails to decrease spine density. Together, these results suggest that alpha1-chimerin is able to modulate dendritic spine morphology by binding to synaptic NMDA receptors and locally inactivating Rac1.

Animals↗
PubMed2005

K channel subconductance levels result from heteromeric pore conformations.

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (K(v)2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the "open" state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open-close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

Allosteric Regulation↗
PubMed2005

Translation of an integral membrane protein in distal dendrites of hippocampal neurons.

Maintenance of synaptic plasticity requires protein translation. Because changes in synaptic strength are regulated at the level of individual synapses, a mechanism is required for newly translated proteins to specifically and persistently modify only a subset of synapses. Evidence suggests this may be accomplished through local translation of proteins at or near synapses in response to plasticity-inducing patterns of activity. A number of proteins important for synaptic function are integral membrane proteins, which require a specialized group of organelles, proteins and enzymatic activities for proper synthesis. Dendrites appear to contain machinery necessary for the proper production of these proteins, and mRNAs for integral membrane proteins have been found localized to dendrites. Experiments are described that investigate the local translation of membrane proteins in the dendrites of cultured rat hippocampal neurons, using fluorescence recovery after photobleaching. Neurons were transfected with cDNAs encoding a fluorescently labeled transmembrane protein, TGN-38. Under conditions where the transport of this reporter construct was inhibited, the appearance of newly synthesized protein was observed via fluorescent microscopy. The dendritic translation of this protein required activation of glutamate receptors. The results demonstrate a functional capacity for activity-dependent synthesis of integral membrane proteins for distal dendrites in hippocampal neurons.

Animals↗
PubMed2004

Rapid dendritic transport of TGN38, a putative cargo receptor.

Protein transport to and from the postsynaptic plasma membrane is thought to be of central importance for synaptic plasticity. However, the molecular details of such processes are poorly understood. One mechanism by which membrane and secretory proteins may be transported to and from postsynaptic membranes is via cargo receptors. We studied the dendritic transport of TGN38, a putative cargo receptor thought to mediate protein transport between the trans-Golgi network (TGN), endosomes, and the plasma membrane. With fluorescence time-lapse imaging of neurons expressing a TGN38-green fluorescent protein fusion protein (GFP-TGN38), we observed rapid bidirectional dynamics of the protein in dendritic shafts. In addition, the protein was present on the surface and on intracellular membranes of dendrites and dendritic spines. Finally, GFP-TGN38 was found to cycle rapidly between the plasma membrane and intracellular membranes within dendrites, including those of spines. Together, our results suggest a role for TGN38 in facilitating rapid changes in the protein composition of postsynaptic membranes.

Animals↗
PubMed2004

The identification of a second actin-binding region in spinophilin/neurabin II.

Spinophilin/neurabin II is an actin-associated scaffolding protein enriched in the dendritic spines of neurons. Previously, the actin-binding domain (ABD) of spinophilin was localized to a domain between amino acids (aa) 1 and 154. In a mass spectrometry screen for spinophilin-binding proteins, we have identified an additional actin-binding region between aa 151 and 282. F-actin co-sedimentation and GST affinity chromotography experiments further substantiate this result. Phalloidin staining of Rat2 fibroblasts transiently expressing GFP-spinophilin deletion constructs indicates co-localization with a subset of actin. Regions of spinophilin that lack the revised ABD (aa 1-230) do not co-localize with phalloidin-labeled actin, suggesting that the actin-binding domain contributes to directing the subcellular distribution of spinophilin. Targeting experiments using primary hippocampal cultures indicate that only the first actin-binding site contributes to dendritic spine localization. The second ABD targets to spines inefficiently and thus may interact with and affect actin filaments in a different manner than the first ABD.

Actin Cytoskeleton↗
PubMed2004

K channel gating by an affinity-switching selectivity filter.

A universal property of ion channels is their ability to alternate stochastically between two permeation states, open and closed. This behavior is thought to be controlled by a steric "gate", a structure that physically impedes ion flow in the closed state and moves out of the way during channel opening. Experiments employing macroscopic currents in the Shaker K channel have suggested a cytoplasmic localization for the gate. Crystallographic structures of the KcsA K channel indeed reveal a cytoplasmic constriction, implying that the gate and selectivity filter are localized to opposite ends of the permeation pathway. However, analysis of K channel subconductance behavior has suggested a strict coupling between channel opening (gating) and permeation. The idea that the selectivity filter is the gate was therefore investigated by using Monte Carlo simulations. Gating is accomplished by allowing the filter to alternate stochastically between two conformations: a high-affinity state, which selectively binds K ions (but not Na ions) and traps them, and a completely nonselective, low-affinity state, which allows both Na and K ions to permeate. The results of these simulations indicate that affinity switching not only endows the selectivity filter with gating abilities, it also allows efficient permeation without jeopardizing ion selectivity. In this model, permeation and gating result from the same process.

Algorithms↗
PubMed2004

Ligand-binding residues integrate affinity and efficacy in the NMDA receptor.

The interaction of an agonist with its receptor can be characterized by two fundamental properties, affinity and efficacy. Affinity defines how tightly the agonist associates with its receptor, and efficacy measures the ability of the bound ligand to activate the receptor. Although affinity and efficacy are independent properties, the binding and activation processes that they describe are tightly coupled. This strong coupling has complicated the interpretation of concentration-response phenotypes caused by receptor mutations. We present an approach that quantifies the role of individual amino acids in defining affinity and efficacy. This method, which employs partial agonists and covalent modification of introduced cysteines, was applied to the ligand-binding sites of the NMDA receptor. Recent crystallographic structures for glutamate receptor ligand-binding cores allowed identification of residues that are either known or are predicted to be critical for ligand binding in the NR1 and NR2A subunit, respectively. Mutation of amino acids whose sidechains would directly coordinate bound ligands affected both agonist affinity and efficacy. In contrast, positions predicted to stabilize the closed-cleft conformation contributed only to agonist efficacy. The results provide a molecular basis for the tight coupling of agonist binding and receptor activation.

Animals↗
PubMed2002

The NMDA receptor M3 segment is a conserved transduction element coupling ligand binding to channel opening.

Ion channels alternate stochastically between two functional states, open and closed. This gating behavior is controlled by membrane potential or by the binding of neurotransmitters in voltage- and ligand-gated channels, respectively. Although much progress has been made in defining the structure and function of the ligand-binding cores and the voltage sensors, how these domains couple to channel opening remains poorly understood. Here we show that the M3 transmembrane segments of the NMDA receptor allosterically interact with both the ligand-binding cores and the channel gate. It is proposed that M3 functions as a transduction element whose conformational change couples ligand binding with channel opening. Furthermore, amino acid homology between glutamate receptor M3 segments and the equivalent S6 or TM2 segments in K(+) channels suggests that ion channel activation and gating are both structurally and functionally conserved.

Amino Acid Motifs↗
PubMedDate not supplied

Effects of mRNA untranslated regions on translational efficiency of NMDA receptor subunits.

Because NMDA receptors play critical roles in both neuronal survival and plasticity, their expression levels need to be carefully controlled. The number of functional NMDA receptors is temporally and spatially regulated at a hierarchy of levels, from gene transcription to protein trafficking. In this review we will focus on mechanisms for controlling functional expression of NMDA receptors that involve altering the efficacy of mRNA translation. One advantage of this level of control is that new receptors can be generated both rapidly and locally in response to appropriate synaptic activity patterns.

Alternative Splicing↗