Teresa K. Aman

Regulation of Persistent Na Current by Interactions between β Subunits of Voltage-Gated Na Channels

The β subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming α subunits, as well as their trafficking and localization. In heterologous expression systems, β1, β2, and β3 subunits influence inactivation and persistent current in different ways. To test how the β4 protein regulates Na channel gating, we transfected β4 into HEK (human embryonic kidney) cells stably expressing NaV1.1. Unlike a free peptide with a sequence from the β4 cytoplasmic domain, the full-length β4 protein did not block open channels. Instead, β4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of β1 or chimeric subunits including the β1 extracellular domain, however, favored inactivation. Coexpressing NaV1.1 and β4 with β1 produced tiny persistent currents, indicating that β1 overcomes the effects of β4 in heterotrimeric channels. In contrast, β1C121W, which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by β4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with β4, persistent current was slightly but significantly increased. Moreover, in β4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that β1 and β4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted β1 subunits.

D2-Like Dopamine Receptors Depolarize Dorsal Raphe Serotonin Neurons through the Activation of Nonselective Cationic Conductance

The dorsal raphe (DR) receives a prominent dopamine (DA) input that has been suggested to play a key role in the regulation of central serotoninergic transmission. DA is known to directly depolarize DR serotonin neurons, but the underlying mechanisms are not well understood. Here, we show that activation of D2-like dopamine receptors on DR 5-HT neurons elicits a membrane depolarization and an inward current associated with an increase in membrane conductance. The DA-induced inward current (IDA) exhibits a linear I-V relationship and reverses polarity at around –15 mV, suggesting the involvement of a mixed cationic conductance. Consistent with this notion, lowering the extracellular concentration of sodium reduces the amplitude of IDA and induces a negative shift of its reversal potential to approximately –45 mV. This current is abolished by inhibiting G-protein function with GDPβS. Examination of the downstream signaling mechanisms reveals that activation of the nonselective cation current requires the stimulation of phospholipase C but not an increase in intracellular calcium. Thus, pharmacological inhibition of phospholipase C reduces the amplitude of IDA. In contrast, buffering intracellular calcium has no effect on the amplitude of IDA. Bath application of transient receptor potential (TRP) channels blockers, 2-aminoethoxydiphenyl borate and SKF96365 [1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole], strongly inhibits IDA amplitude, suggesting the involvement of TRP-like conductance. These results reveal previously unsuspected mechanism by which D2-like DA receptors induce membrane depolarization and enhance the excitability of DR 5-HT neurons.

Phosphorylation of Ser1166 on GluN2B by PKA Is Critical to Synaptic NMDA Receptor Function and Ca2+ Signaling in Spines

The NMDA-type glutamate receptor (NMDAR) is essential for synaptogenesis, synaptic plasticity, and higher cognitive function. Emerging evidence indicates that NMDAR Ca2+ permeability is under the control of cAMP/protein kinase A (PKA) signaling. Whereas the functional impact of PKA on NMDAR-dependent Ca2+ signaling is well established, the molecular target remains unknown. Here we identify serine residue 1166 (Ser1166) in the carboxy-terminal tail of the NMDAR subunit GluN2B to be a direct molecular and functional target of PKA phosphorylation critical to NMDAR-dependent Ca2+ permeation and Ca2+ signaling in spines. Activation of β-adrenergic and D1/D5-dopamine receptors induces Ser1166 phosphorylation. Loss of this single phosphorylation site abolishes PKA-dependent potentiation of NMDAR Ca2+ permeation, synaptic currents, and Ca2+ rises in dendritic spines. We further show that adverse experience in the form of forced swim, but not exposure to fox urine, elicits striking phosphorylation of Ser1166 in vivo, indicating differential impact of different forms of stress. Our data identify a novel molecular and functional target of PKA essential to NMDAR-mediated Ca2+ signaling at synapses and regulated by the emotional response to stress.

C-terminal Domains of N-Methyl-d-aspartic Acid Receptor Modulate Unitary Channel Conductance and Gating

Background: The C-terminal domains (CTDs) of NMDA receptors are essential for normal brain function. Results: We developed kinetic mechanisms for receptors lacking CTDs using single-channel methods. Conclusion: GluN1 CTDs control primarily unitary conductance and GluN2 CTDs control gating kinetics. Significance: Results afford quantitative insight into how intracellular perturbations can change the time course of NMDA receptor currents. NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor.

Inwardly Permeating Na Ions Generate the Voltage Dependence of Resurgent Na Current in Cerebellar Purkinje Neurons

Voltage-gated Na channels of cerebellar Purkinje neurons express an endogenous open-channel blocking protein. This blocker binds channels at positive potentials and unbinds at negative potentials, generating a resurgent Na current and permitting rapid firing. The macroscopic voltage dependence of resurgent current raises the question of whether the blocker directly senses membrane potential or whether voltage dependence is conferred indirectly. Because we previously found that inwardly permeating Na ions facilitate dissociation of the blocker, we measured voltage-clamped currents in different Na gradients to test the role of permeating ions in generating the voltage dependence of unblock. In reverse gradients, outward resurgent currents were tiny or absent, suggesting that unblock normally requires “knockoff” by Na. Inward resurgent currents at strongly negative potentials, however, were larger in reverse than in control gradients. Moreover, occupancy of the blocked state was prolonged both in reverse gradients and in control gradients with reduced Na concentrations, indicating that block is more stable when inward currents are small. Accordingly, reverse gradients shifted the voltage dependence of block, such that resurgent currents were evoked even after conditioning at negative potentials. Additionally, in control gradients, peak resurgent currents decreased linearly with driving force during the conditioning step, suggesting that the stability of block varies directly with inward Na current amplitude. Thus, the voltage dependence of blocker unbinding results almost entirely from repulsion by Na ions occupying the external pore. The lack of voltage sensitivity of the blocking protein suggests that the blockers binding site lies outside the membrane field, in the permeation pathway.

Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability

Background: PKA increases NMDA receptor responses and phosphorylates multiple residues on C-terminal domains (CTD). Results: PKA inhibition reduced gating through GluN2B CTD and reduced Ca2+ permeability through GluN1 CTD. Conclusion: PKA controls NMDA receptor gating and Ca2+ permeability through distinct sites. Significance: Dissecting the complex modulatory effects of PKA on NMDA receptors helps delineate fundamental mechanisms of synaptic regulation. Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca2+ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca2+ permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca2+ conductance, because neither Na+ conductance nor Ca2+-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca2+ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca2+ permeability.

Antagonism of Lidocaine Inhibition by Open-Channel Blockers That Generate Resurgent Na Current

Na channels that generate resurgent current express an intracellular endogenous open-channel blocking protein, whose rapid binding upon depolarization and unbinding upon repolarization minimizes fast and slow inactivation. Na channels also bind exogenous compounds, such as lidocaine, which functionally stabilize inactivation. Like the endogenous blocking protein, these use-dependent inhibitors bind most effectively at depolarized potentials, raising the question of how lidocaine-like compounds affect neurons with resurgent Na current. We therefore recorded lidocaine inhibition of voltage-clamped, tetrodotoxin-sensitive Na currents in mouse Purkinje neurons, which express a native blocking protein, and in mouse hippocampal CA3 pyramidal neurons with and without a peptide from the cytoplasmic tail of NaVβ4 (the β4 peptide), which mimics endogenous open-channel block. To control channel states during drug exposure, lidocaine was applied with rapid-solution exchange techniques during steps to specific voltages. Inhibition of Na currents by lidocaine was diminished by either the β4 peptide or the native blocking protein. In peptide-free CA3 cells, prolonging channel opening with a site-3 toxin, anemone toxin II, reduced lidocaine inhibition; this effect was largely occluded by open-channel blockers, suggesting that lidocaine binding is favored by inactivation but prevented by open-channel block. In constant 100 μm lidocaine, current-clamped Purkinje cells continued to fire spontaneously. Similarly, the β4 peptide reduced lidocaine-dependent suppression of spiking in CA3 neurons in slices. Thus, the open-channel blocking protein responsible for resurgent current acts as a natural antagonist of lidocaine. Neurons with resurgent current may therefore be less susceptible to use-dependent Na channel inhibitors used as local anesthetic, antiarrhythmic, and anticonvulsant drugs.