Anastassios V. Tzingounis

Glutamate transporters: confining runaway excitation by shaping synaptic transmission

Traditionally, glutamate transporters have been viewed as membrane proteins that harness the electrochemical gradient to slowly transport glutamate from the extracellular space into glial cells. However, recent studies have shown that glutamate transporters on glial and neuronal membranes also rapidly bind released glutamate to shape synaptic transmission. In this Review, we summarize the properties of glutamate transporters that influence synaptic transmission and are subject to regulation and plasticity. We highlight how the diversity of glutamate-transporter function relates to transporter location, density and affinity.

Arc/Arg3.1: Linking Gene Expression to Synaptic Plasticity and Memory

Arc/Arg3.1 is an effector immediate-early gene implicated in the consolidation of memories. Although cloned a decade ago, the physiological role of Arc/Arg3.1 in the brain has remained elusive. Four papers in this issue of Neuron address this function. These studies show that Arc/Arg3.1 regulates endophilin 3 and dynamin 2, two components of the endocytosis machinery. Genetic ablation of Arc/Arg3.1 in mice or overexpression in culture suggest that Arc/Arg3.1 regulates AMPA receptor trafficking and synaptic plasticity. Finally, Arc/Arg3.1 knockout mice show memory retention deficits. These recent developments provide new insights into the function of this popular activity-dependent neuronal marker.

Molecular constituents of neuronal AMPA receptors

Dynamic regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) underlies aspects of synaptic plasticity. Although numerous AMPAR-interacting proteins have been identified, their quantitative and relative contributions to native AMPAR complexes remain unclear. Here, we quantitated protein interactions with neuronal AMPARs by immunoprecipitation from brain extracts. We found that stargazin-like transmembrane AMPAR regulatory proteins (TARPs) copurified with neuronal AMPARs, but we found negligible binding to GRIP, PICK1, NSF, or SAP-97. To facilitate purification of neuronal AMPAR complexes, we generated a transgenic mouse expressing an epitope-tagged GluR2 subunit of AMPARs. Taking advantage of this powerful new tool, we isolated two populations of GluR2 containing AMPARs: an immature complex with the endoplasmic reticulum chaperone immunoglobulin-binding protein and a mature complex containing GluR1, TARPs, and PSD-95. These studies establish TARPs as the auxiliary components of neuronal AMPARs.

Contribution of KCNQ2 and KCNQ3 to the medium and slow afterhyperpolarization currents

Benign familial neonatal convulsion (BNFC) is a neurological disorder caused by mutations in the potassium channel genes KCNQ2 and KCNQ3, which are thought to contribute to the medium afterhyperpolarization (mAHP). Despite their importance in normal brain function, it is unknown whether they invariably function as heteromeric complexes. Here, we examined the contribution of KCNQ3 and KCNQ2 in mediating the apamin-insensitive mAHP current (ImAHP) in hippocampus. The ImAHP was not impaired in CA1 pyramidal neurons from mice genetically deficient for either KCNQ3 or KCNQ2 but was reduced ≈50% in dentate granule cells. While recording from KCNQ-deficient mice, we observed that the calcium-activated slow afterhyperpolarization current (IsAHP) was also reduced in dentate granule cells, suggesting that KCNQ channels might also contribute to this potassium current whose molecular identity is unknown. Further pharmacological and molecular experiments manipulating KCNQ channels provided evidence in support of this possibility. Together our data suggest that multiple KCNQ subunit compositions can mediate the ImAHP, and that the very same subunits may also contribute to the IsAHP. We also present data suggesting that the neuronal calcium sensor protein hippocalcin may allow for these dual signaling processes.

Hippocalcin Gates the Calcium Activation of the Slow Afterhyperpolarization in Hippocampal Pyramidal Cells

In the brain, calcium influx following a train of action potentials activates potassium channels that mediate a slow afterhyperpolarization current (I(sAHP)). The key steps between calcium influx and potassium channel activation remain unknown. Here we report that the key intermediate between calcium and the sAHP channels is the diffusible calcium sensor hippocalcin. Brief depolarizations sufficient to activate the I(sAHP) in wild-type mice do not elicit the I(sAHP) in hippocalcin knockout mice. Introduction of hippocalcin in cultured hippocampal neurons leads to a pronounced I(sAHP), while neurons expressing a hippocalcin mutant lacking N-terminal myristoylation exhibit a small I(sAHP) that is similar to that recorded in uninfected neurons. This implies that hippocalcin must bind to the plasma membrane to mediate its effects. These findings support a model in which the calcium sensor for the sAHP channels is not preassociated with the channel complex.

The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus

Mutations in KCNQ2 and KCNQ3 voltage-gated potassium channels lead to neonatal epilepsy as a consequence of their key role in regulating neuronal excitability. Previous studies in the brain have focused primarily on these KCNQ family members, which contribute to M-currents and afterhyperpolarization conductances in multiple brain areas. In contrast, the function of KCNQ5 (Kv7.5), which also displays widespread expression in the brain, is entirely unknown. Here, we developed mice that carry a dominant negative mutation in the KCNQ5 pore to probe whether it has a similar function as other KCNQ channels. This mutation renders KCNQ5dn-containing homomeric and heteromeric channels nonfunctional. We find that Kcnq5dn/dn mice are viable and have normal brain morphology. Furthermore, expression and neuronal localization of KCNQ2 and KCNQ3 subunits are unchanged. However, in the CA3 area of hippocampus, a region that highly expresses KCNQ5 channels, the medium and slow afterhyperpolarization currents are significantly reduced. In contrast, neither current is affected in the CA1 area of the hippocampus, a region with low KCNQ5 expression. Our results demonstrate that KCNQ5 channels contribute to the afterhyperpolarization currents in hippocampus in a cell type-specific manner.

β-Blocker drugs mediate calcium signaling in native central nervous system neurons by β-arrestin–biased agonism

G protein-coupled receptors (GPCRs), the largest family of signaling receptors expressed in the CNS, mediate the neuropsychiatric effects of a diverse range of clinically relevant drugs. It is increasingly clear that GPCRs can activate distinct G protein-dependent and -independent transduction pathway(s), and that certain drugs differ in the ability to regulate distinct signaling mechanisms linked to the same receptors. A fundamental question in neuropharmacology is whether such “biased agonism” occurs in physiologically relevant neurons and with endogenous receptors. Here we show that propranolol and carvedilol, two β-blocker drugs that inhibit β-adrenergic signaling via heterotrimeric G proteins, function in hippocampal pyramidal neurons as potent and selective activators of an alternate receptor-linked calcium signaling pathway mediated by β-arrestin-2 and ERK1/2. Our results support the emerging view of β-arrestin–biased agonism as a significant mechanism of drug action and do so in CNS-derived neurons expressing only native receptors.

Presynaptic NMDA receptors get into the act

In a new form of synaptic plasticity, depolarization of Purkinje cells increases the frequency of mini GABAergic IPSCs, via glutamate acting as a retrograde messenger on presynaptic NMDA receptors on the terminals of cerebellar interneurons.