Acid-sensing ion channels (ASICs), a novel class of ligand-gated cation channels activated by protons, are highly expressed in peripheral sensory and central neurons. Activation of ASICs may play an important role in physiological processes such as nociception, mechanosensation, and learning-memory, and in the pathology of neurological conditions such as brain ischemia. Modulation of the activities of ASICs is expected to have a significant influence on the roles that these channels can play in both physiological and/or pathological processes. Here we show that the divalent cation Zn²⁺, an endogenous trace element, dose-dependently inhibits ASIC currents in cultured mouse cortical neurons at nanomolar concentrations. With ASICs expressed in Chinese hamster ovary cells, Zn²⁺ inhibits currents mediated by homomeric ASIC1a and heteromeric ASIC1a-ASIC2a channels, without affecting currents mediated by homomeric ASIC1β, ASIC2a, or ASIC3. Consistent with ASIC1a-specific modulation, high-affinity Zn²⁺ inhibition is absent in neurons from ASIC1a knock-out mice. Current-clamp recordings and Ca²⁺-imaging experiments demonstrated that Zn²⁺ inhibits acid-induced membrane depolarization and the increase of intracellular Ca²⁺. Mutation of lysine-133 in the extracellular domain of the ASIC1a subunit abolishes the high-affinity Zn²⁺ inhibition. Our studies suggest that Zn²⁺ may play an important role in a negative feedback system for preventing overexcitation of neurons during normal synaptic transmission and ASIC1a-mediated excitotoxicity in pathological conditions.

Ischemic brain injury is a major problem associated with stroke. It has been increasingly recognized that acid-sensing ion channels (ASICs) contribute significantly to ischemic neuronal damage, but the underlying mechanism has remained elusive. Here, we show that extracellular spermine, one of the endogenous polyamines, exacerbates ischemic neuronal injury through sensitization of ASIC1a channels to extracellular acidosis. Pharmacological blockade of ASIC1a or deletion of theASIC1gene greatly reduces the enhancing effect of spermine in ischemic neuronal damage both in cultures of dissociated neurons and in a mouse model of focal ischemia. Mechanistically, spermine profoundly reduces desensitization of ASIC1a by slowing down desensitization in the open state, shifting steady-state desensitization to more acidic pH, and accelerating recovery between repeated periods of acid stimulation. Spermine-mediated potentiation of ASIC1a activity is occluded by PcTX1 (psalmotoxin 1), a specific ASIC1a inhibitor binding to its extracellular domain. Functionally, the enhanced channel activity is accompanied by increased acid-induced neuronal membrane depolarization and cytoplasmic Ca²⁺overload, which may partially explain the exacerbated neuronal damage caused by spermine. More importantly, blocking endogenous spermine synthesis significantly attenuates ischemic brain injury mediated by ASIC1a but not that by NMDA receptors. Thus, extracellular spermine contributes significantly to ischemic neuronal injury through enhancing ASIC1a activity. Our data suggest new neuroprotective strategies for stroke patients via inhibition of polyamine synthesis and subsequent spermine–ASIC interaction.

Ion channels are involved in normal physiologic processes and in the pathology of various diseases. In this study, we investigated the presence and potential function of transient receptor potential melastatin 7 (TRPM7) channels in the growth and proliferation of FaDu and SCC25 cells, two common human head and neck squamous carcinoma cell lines, using a combination of patch-clamp recording, Western blotting, immunocytochemistry, small interfering RNA (siRNA), fluorescent Ca²⁺ imaging, and cell counting techniques. Although voltage-gated K⁺ currents were recorded in all cells, none of FaDu cells express voltage-gated Na⁺ or Ca²⁺ currents. Perfusion of cells with NMDA or acidic solution did not activate inward currents, indicating a lack of NMDA receptor and acid-sensing channels. Lowering extracellular Ca²⁺, however, induced a large nondesensitizing current reminiscent of Ca²⁺-sensing cation current or TRPM7 current previously described in other cells. This Ca²⁺-sensing current can be inhibited by Gd³⁺, 2-aminoethoxydiphenyl borate (2-APB), or intracellular Mg²⁺, consistent with the TRPM7 current being activated. Immunocytochemistry, Western blot, and reverse transcription-PCR detected the expression of TRPM7 protein and mRNA in these cells. Transfection of FaDu cells with TRPM7 siRNA significantly reduced the expression of TRPM7 mRNA and protein as well as the amplitude of the Ca²⁺-sensing current. Furthermore, we found that Ca²⁺ is critical for the growth and proliferation of FaDu cells. Blockade of TRPM7 channels by Gd³⁺ and 2-APB or suppression of TRPM7 expression by siRNA inhibited the growth and proliferation of these cells. Similar to FaDu cells, SCC25 cells also express TRPM7-like channels. Suppressing the function of these channels inhibited the proliferation of SCC25 cells. [Cancer Res 2007;67(22):10929–38]