Antonius M. J. VanDongen

Alteration and restoration of K+ channel function by deletions at the N- and C-termini

Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.

Fast and slow gating of sodium channels encoded by a single mRNA

We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein.

Activation-Dependent Subconductance Levels in the drk1 K Channel Suggest a Subunit Basis for Ion Permeation and Gating

Ion permeation and channel opening are two fundamental properties of ion channels, the molecular bases of which are poorly understood. Channels can exist in two permeability states, open and closed. The relative amount of time a channel spends in the open conformation depends on the state of activation. In voltage-gated ion channels, activation involves movement of a charged voltage sensor, which is required for channel opening. Single-channel recordings of drk1 K channels expressed in Xenopus oocytes suggested that intermediate current levels (sublevels) may be associated with transitions between the closed and open states. Because K channels are formed by four identical subunits, each contributing to the lining of the pore, it was hypothesized that these sublevels resulted from heteromeric pore conformations. A formal model based on this hypothesis predicted that sublevels should be more frequently observed in partially activated channels, in which some but not all subunits have undergone voltage-dependent conformational changes required for channel opening. Experiments using the drk1 K channel, as well as drk1 channels with mutations in the pore and in the voltage sensor, showed that the probability of visiting a sublevel correlated with voltage- and time-dependent changes in activation. A subunit basis is proposed for channel opening and permeation in which these processes are coupled.

Valproate Reduces Excitability by Blockage of Sodium and Potassium Conductance

Summary: Effects of the antiepileptic drug valproate on sodium and potassium currents in the nodal membrane of peripheral nerve fibers of Xenopus laevis were determined by voltage‐ and current‐clamp experiments. Under voltage‐clamp conditions, a reduction of both sodium and potassium conductance (in a ratio of 2:1) was observed. Typically, 2.4 mM (400 mg/L) valproate reduced the sodium current 54% and the potassium current 26%, at a membrane potential of 5 mV. Valproate did not affect the leakage conductance. The reduction of potassium conductance was voltage dependent, being more pronounced at more positive membrane potentials. For the sodium system, a voltage dependency of the blockage could not be established. Under current‐clamp conditions, valproate caused a reduction of excitability of nerve membrane: amplitude of the action potential and maximum rate of rise were decreased, whereas threshold potential was increased. The ability to follow high‐frequency stimulation was impaired.

The Nonkinase Phorbol Ester Receptor α1-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 α1-chimerin is present in dendrites and spines, where it binds to the NMDA receptor NR2A subunit in a phorbol ester-dependent manner. α1-Chimerin contains a GTPase activating (GAP) domain, with activity toward the Rho family member Rac1. Overexpression of α1-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 α1-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 α1-chimerin to modulate spine number requires an interaction with the NMDA receptor, because an α1-chimerin mutant that binds weakly to NR2A fails to decrease spine density. Together, these results suggest that α1-chimerin is able to modulate dendritic spine morphology by binding to synaptic NMDA receptors and locally inactivating Rac1.

Nuclear deformation during breast cancer cell transmigration.

Metastasis is the main cause of cancer mortality. During this process, cancer cells dislodge from a primary tumor, enter the circulation and form secondary tumors in distal organs. It is poorly understood how these cells manage to cross the tight syncytium of endothelial cells that lines the capillaries. Such capillary transmigration would require a drastic change in cell shape. We have therefore developed a microfluidic platform to study the transmigration of cancer cells. The device consists of an array of microchannels mimicking the confined spaces encountered. A thin glass coverslip bottom allows high resolution imaging of cell dynamics. We show that nuclear deformation is a critical and rate-limiting step for transmigration of highly metastatic human breast cancer cells. Transmigration was significantly reduced following the treatment with a protein methyltransferase inhibitor, suggesting that chromatin condensation might play an important role. Since transmigration is critical for cancer metastasis, this new platform may be useful for developing improved cancer therapies.

Arc/Arg3.1 Translation Is Controlled by Convergent N-Methyl-D-aspartate and Gs-coupled Receptor Signaling Pathways

Arc/Arg3.1 is an immediate early gene whose expression is necessary for the late-phase of long-term potentiation (LTP) and memory consolidation. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. Fluorescence microscopy experiments in cultured hippocampal neurons revealed that Arc protein level was dramatically increased by activation of the cAMP-dependent protein kinase (PKA) pathway, which is implicated in long-term memory. A PKA-dependent increase in Arc protein level was observed after pharmacological or synaptic activation of N-methyl-d-aspartate (NMDA) receptors, which play a critical role in both LTP induction and learning. Arc protein was also up-regulated by activation of PKA through Gs-coupled dopamine and β-adrenergic receptors, which regulate the late-phase of LTP and memory. When agonists for the NMDA and Gs-coupled receptors were co-applied, they had an additive effect on Arc protein expression. Interestingly, Gs-coupled receptor stimulation was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect Arc mRNA level or protein stability, identifying translational efficacy as the main determinant of Arc protein expression level. It is concluded that efficient Arc translation requires NMDA receptor activity, whereas a further enhancement can be achieved with activation of Gs-coupled receptors. These experiments have, therefore, revealed remarkable similarities in the signaling pathways that control Arc expression and those that regulate LTP, learning, and memory.

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 (Kv2.1) 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.

Biology of the NMDA Receptor

NMDA Receptors and Brain Development, R.C. Ewald and H.T. Cline NMDA Receptors and Huntingtons Disease, H.B. Fernandes and L.A. Raymond NMDA and Dopamine Diverse Mechanisms Applied to Interacting Receptor Systems, C. Cepeda, V.M. Andre, E.L. Jocoy, and M.S. Levine NMDA Receptors and Alcohol Addiction, D. Ron and J. Wang Transcriptional Regulation of NMDAR Expression, G. Bai and P.W. Hoffman NMDARs and Translational Control, C.A. Hoeffer and E. Klann Regulation of NMDARs by Kinases and Phosphatases, M.W. Salter, Y. Dong, L.V. Kalia, X.J. Liu, and G. Pitcher Trafficking and Targeting of NMDARs, R.S. Petralia, R.A. Al-Hallaq, and R.J. Wenthold NMDAR-Mediated Calcium Transients in Dendritic Spines, B.L. Bloodgood and B.L. Sabatini NMDA Receptors in Drosophila, S. Xia and A.-S. Chiang Extracellular Modulation of NMDARs, K. Williams Pharmacology of NMDARs, D.T. Monaghan and D.E Jane Activation Mechanisms of NMDARs, M.L. Blanke and A M.J. VanDongen Presynaptic NMDARs, I.C. Duguid and T.G. Smart

Localization and Phosphorylation of Abl-Interactor Proteins, Abi-1 and Abi-2, in the Developing Nervous System

Abl-interactor (Abi) proteins are targets of Abl-family nonreceptor tyrosine kinases and are required for Rac-dependent cytoskeletal reorganization in response to growth factor stimulation. We asked if the expression, phosphorylation, and cellular localization of Abi-1 and Abi-2 supports a role for these proteins in Abl signaling in the developing and adult mouse nervous system. In mid- to late-gestation embryos, abi-2 message is elevated in the central and peripheral nervous systems (CNS and PNS). Abi-1 mRNA is present, but not enhanced, in the CNS, and is not observed in PNS structures. Abi proteins from brain lysates undergo changes in apparent molecular weight and phosphorylation with increasing age. In the postnatal brain, abi-1 and abi-2 are expressed most prominently in cortical layers populated by projection neurons. In cultured neurons, Abi-1 and Abi-2 are concentrated in puncta throughout the cell body and processes. Both Abi and Abl proteins are present in synaptosomes and growth cone particles. Therefore, the Abi adaptors exhibit proper expression patterns and subcellular localization to participate in Abl kinase signaling in the nervous system.