PSD-95 and Lin-7b Interact with Acid-sensing Ion Channel-3 and Have Opposite Effects on H+-gated Current

Abstract

The acid-sensing ion channel-3 (ASIC3) is a degenerin/ epithelial sodium channel expressed in the peripheral nervous system. Previous studies indicate that it participates in the response to mechanical and painful stimuli, perhaps contributing to mechanoreceptor and/or H ؉ -gated nociceptor function. ASIC3 subunits contain intracellular N and C termini that may control channel localization and function. We found that a PDZ-binding motif at the ASIC3 C terminus interacts with four different proteins that contain PDZ domains: PSD-95, Lin7b, MAGI-1b, and PIST. ASIC3 and these interacting proteins were expressed in dorsal root ganglia and spinal cord, and PSD-95 co-precipitated ASIC3 from spinal cord. When expressed in heterologous cells, PSD-95 reduced the amplitude of ASIC3 acid-evoked currents, whereas Lin-7b increased current amplitude. PSD-95 and Lin-7b altered current density by decreasing or increasing, respectively, the amount of ASIC3 on the cell surface. The finding that multiple PDZ-containing proteins bind ASIC3 and can influence its presence in the plasma membrane suggests that they may play an important role in the contribution of ASIC3 to nociception and mechanosensation. ASIC31 is a non-voltage-gated Na ϩ channel activated by acidic extracellular solutions (1, 2). It is expressed in the peripheral nervous system, including the dorsal root ganglia and trigeminal ganglia. Immunocytochemical studies have localized it to several different specialized sensory nerve endings of skin, suggesting it might participate in mechanosensation and nociception (3). The importance of ASIC3 for sensory function was revealed by studies of mice bearing targeted disruptions of the ASIC3 gene (3, 4). In single fiber recordings from cutaneous nerves, loss of ASIC3 increased the sensitivity of mechanoreceptors detecting light touch but reduced the sensitivity of a mechanoreceptor responding to noxious pinch. In behavioral studies, ASIC3 modulated the response to noxious acid, heat, and mechanical stimuli. Moreover chronic mechanical hyperalgesia produced by intramuscular acid injections was prevented in ASIC3 null animals (5). Functional studies suggest that ASIC3 may also mediate the pain associated with myocardial ischemia (6, 7). Thus, in different cellular contexts, ASIC3 may participate in the responses to both mechanical and acidotic stimuli to mediate normal touch and pain sensation.ASIC3, a member of the DEG/ENaC family, forms heteromultimers in neurons with other DEG/ENaC subunits, ASIC1 and ASIC2, to generate H ϩ -gated cation channels (8 -10). ASIC3 shares the overall structure of DEG/ENaC proteins, including two transmembrane domains, a large extracellular loop containing 14 conserved cysteines, and intracellular N and C termini (11,12). The intracellular domains of ASIC subunits might have several functions, including localizing the channels, controlling the number of channels on the cell surface, regulating channel function, and forming part of a scaffold important for the response to mechanical stimuli (11-13)...

Acid-sensing ion channel 3 (ASIC3) cell surface expression is modulated by PSD-95 within lipid rafts

“…Rat ASIC3 in pMT3 vector was cloned as described previously (20). HA-ASIC3 was generated by insertion of two hemagglutinin (HA) epitopes (YPYDVPDYA-G-YPYDVPDYA) at the NH2 terminus of ASIC3.…”

“…Knockdown of PSD-95 in rat spinal cord attenuated, and targeted disruption of PSD-95 in mice abolished, hyperalgesia to mechanical and thermal stimuli following nerve injury (15,43,44). Previously, work in our laboratory demonstrated that both PSD-95 and ASIC3 are present in dorsal root ganglia and coimmunoprecipitate together in rat spinal cord (20).PSD-95 is essential for normal synaptic plasticity at postsynaptic sites, where it integrates signaling by localizing and clustering proteins (26). PSD-95 forms multimers, and each subunit contains three PDZ domains.…”

“…The mechanism appears to involve trafficking of ASIC3: Lin-7b increased and PSD-95 decreased ASIC3 protein expression at the cell surface (20). CIPP (channel-interacting PDZ domain protein), PIST (PDZ protein interacting specifically with TC10), MAGI (membrane-associated guanylate kinase with inverted orientation), and NHERF (Na ϩ /H ϩ exchanger regulatory factor-1) are other PDZ domain-containing proteins that have been shown to interact with ASIC3 and modulate its function (1,11,20).…”

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

“…Although similar to glutamate receptors, they are also present on dendrites and cell bodies (20,(22)(23)(24). ASIC subunits interact with postsynaptic scaffolding proteins, including postsynaptic density protein 95 and protein interacting with C-kinase-1 (20,(24)(25)(26)(27)(28)(29). In addition, ASICs are enriched in synaptosome-containing brain fractions (20,24,30).…”

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

“…The extracellular domain senses protons and interacts with modulators, including proteases, Zn 2ϩ , Ca 2ϩ , and redox reagents (42). The cytoplasmic NH 2 and COOH termini contain phosphorylation sites and interact with other proteins such as protein interacting with C kinase 1 (PICK1) (14,25,42), postsynaptic density protein 95, abnormal cell lineage 7b (24), and annexin (13), resulting in changes in the current density or cellular localization of ASIC. For example, PICK1 increases the ASIC2a current amplitude by potentiating the phosphorylation of ASIC2a at T39 [equivalent to site S40 on human (h)ASIC1b] (3).…”