Abstract
The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.
Highlights
Acid-sensing ion channels (ASICs)2 are Hϩ-gated members of the DEG/epithelial Naϩ channel (ENaC) family [1,2,3]
Reducing ␣-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that ␣-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity
The sequence was not present in ASIC2 or ASIC3, suggesting that ␣-actinin might interact with ASIC1
Summary
Expression Constructs—Mouse ASIC1a was cloned into pMT3 [44] for heterologous expression. To test for efficacy and specificity of siRNA, CHO cells were co-transfected with either one of the siRNAs and EGFP-tagged ␣-actinin at a 10:1 cDNA ratio (total 11 g DNA) using Lipofectamine 2000 in Opti-MEM I media according to the manufacturer’s recommendation (Invitrogen). Electrophysiology—Whole-cell, patch clamp recordings were performed on CHO cells on 10-mm glass coverslips continuously superfused with a bath solution containing: 128 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM MES, 5.55 mM glucose, adjusted to pH 7.4 with TMAOH. For whole-cell, patch clamp experiments with hippocampal neurons the bath solution contained 100 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM MES, 10 mM glucose adjusted to pH 7.4 with TMAOH. Statistical differences were determined by twotailed Student’s t test
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