Acid-sensing ion channels (ASICs) have been implicated in a wide variety of physiological functions. We have used a rat dorsal root ganglion cDNA library in a yeast two-hybrid assay to identify sensory neuron proteins that interact with ASICs. We found that annexin II light chain p11 physically interacts with the N terminus of ASIC1a, but not other ASIC isoforms. Immunoprecipitation studies confirmed an interaction between p11 and ASIC1 in rat dorsal root ganglion neurons in vivo. Coexpression of p11 and ASIC1a in CHO-K1 cells led to a 2-fold increase in expression of the ion channel at the cell membrane as determined by membrane-associated immunoreactivity and cell-surface biotinylation. Consistent with these findings, peak ASIC1a currents in transfected CHO-K1 cells were up-regulated 2-fold in the presence of p11, whereas ASIC3-mediated currents were unaffected by p11 expression. Neither the pH dependence of activation nor the rates of desensitization were altered by p11, suggesting that its primary role in regulating ASIC1a activity is to enhance cell-surface expression of ASIC1a. These data demonstrate that p11, already known to traffic members of the voltage-gated sodium and potassium channel families as well as transient receptor potential and chloride channels, also plays a selective role in enhancing ASIC1a functional expression.

The essence of neuronal function is to generate outputs in response to synaptic potentials. Synaptic integration at postsynaptic sites determines neuronal outputs in the CNS. Using immunohistochemical and electrophysiological approaches, we first reveal that steroidogenic factor 1 (SF-1) green fluorescent protein (GFP)-positive neurons in the ventromedial nucleus of the hypothalamus express P2X4 subunits that are activated by exogenous ATP. Increased membrane expression of P2X4 channels by using a peptide competing with P2X4 intracellular endocytosis motif enhances neuronal excitability of SF-1 GFP-positive neurons. This increased excitability is inhibited by a P2X receptor antagonist. Furthermore, increased surface P2X4 receptor expression significantly decreases the frequency and the amplitude of GABAergic postsynaptic currents of SF-1 GFPpositive neurons. Co-immunopurification and pulldown assays reveal that P2X4 receptors complex with aminobutyric acid, type A (GABA A ) receptors and demonstrate that two amino acids in the carboxyl tail of the P2X4 subunit are crucial for its physical association with GABA A receptors. Mutation of these two residues prevents the physical association, thereby blocking cross-inhibition between P2X4 and GABA A receptors. Moreover, disruption of the physical coupling using competitive peptides containing the identified motif abolishes current inhibition between P2X4 and GABA A receptors in recombinant system and P2X4 receptor-mediated GABAergic depression in SF-1 GFP-positive neurons. Our present work thus provides evidence for cross-talk between excitatory and inhibitory receptors that appears to be crucial in determining GABAergic synaptic strength at a central synapse.ATP acts at cell surface receptors of two fundamentally distinct types: ligand-gated ion channels (P2X receptors) and G-protein-coupled receptors (P2Y receptors). ATP P2X receptors are widely distributed in excitable and non-excitable cells of vertebrates and are nonselective cation channels (1). Their activation mediates membrane depolarization and calcium influx (2). Among the seven P2X subunits, P2X4 receptors are most widely distributed in the CNS (3). It appears that P2X4-containing receptors are involved in purinergic synaptic transmission at central synapses since they are located at postsynaptic sites in the brain, including the hippocampus and the cerebellum (4). Although there is increasing evidence for their implications in various physiological and pathological conditions (5), the physiological role of P2X receptors at the synaptic level has been poorly defined, at least in part, due to the paucity of purinergic synaptic transmission in the CNS.In the nervous system, ATP appears to be primarily a cotransmitter rather than a principal transmitter (6). ATP is released either with an inhibitory neurotransmitter, GABA 4 (7-10) or with an excitatory neurotransmitter, glutamate in the CNS (4, 11). Recent studies have clearly demonstrated the interactions of P2X receptors with other ligand-gated channels, i...

ASIC4 is a member of the acid-sensing ion channel family that is broadly expressed in the mammalian nervous system, but has no known function. We demonstrate here that transfected ASIC4 is targeted to the plasma membrane in CHO-K1 cells, where it associates with ASIC1a and downregulates exogenous ASIC1a expression. This effect could also be observed on endogenous H+-gated currents in TSA-201 cells and ASIC3 currents in CHO-K1 cells, suggesting a physiological role for ASIC4 in regulating ASIC currents involved in pain mechanisms. Using a yeast two-hybrid assay we found that ASICs interact with proteins involved in diverse functions, including cytoskeletal proteins, enzymes, regulators of endocytosis and G-protein-coupled pathways. ASIC4 is the sole member of this ion channel class to interact strongly with polyubiquitin. The distinct functionally related sets of interacting proteins that bind individual ASICs identified in the yeast two-hybrid screen suggest potential roles for ASICs in a variety of cellular functions.