Proton-sensitive cation channels and ion exchangers in ischemic brain injury: new therapeutic targets for stroke?

Abstract

Ischemic brain injury results from complicated cellular mechanisms. The present therapy for acute ischemic stroke is limited to thrombolysis with the recombinant tissue plasminogen activator (rtPA) and mechanical recanalization. Therefore, a better understanding of ischemic brain injury is needed for the development of more effective therapies. Disruption of ionic homeostasis plays an important role in cell death following cerebral ischemia. Glutamate receptor-mediated ionic imbalance and neurotoxicity have been well established in cerebral ischemia after stroke. However, non-NMDA receptor-dependent mechanisms, involving acid-sensing ion channel 1a (ASIC1a), transient receptor potential melastatin 7 (TRPM7), and Na(+)/H(+) exchanger isoform 1 (NHE1), have recently emerged as important players in the dysregulation of ionic homeostasis in the CNS under ischemic conditions. These H(+)-sensitive channels and/or exchangers are expressed in the majority of cell types of the neurovascular unit. Sustained activation of these proteins causes excessive influx of cations, such as Ca(2+), Na(+), and Zn(2+), and leads to ischemic reperfusion brain injury. In this review, we summarize recent pre-clinical experimental research findings on how these channels/exchangers are regulated in both in vitro and in vivo models of cerebral ischemia. The blockade or transgenic knockdown of these proteins was shown to be neuroprotective in these ischemia models. Taken together, these non-NMDA receptor-dependent mechanisms may serve as novel therapeutic targets for stroke intervention.

Keywords: ASIC; Acidosis; Calcium; NHE1; TRPM7; Zinc.

Figure 1. Pathophysiological changes in the neurovascular unit following cerebral ischemia and the participating non-NMDA proton-sensitive ion channels/exchangers

Cerebral ischemia deprives neurons of energy required to maintain ionic homeostasis. Homomeric acid-sensing ion channel 1a (ASIC1a), transient receptor potential melastatin 7 (TRPM7), and Na⁺/H⁺ exchanger isoform 1 (NHE1) are proton-sensitive channels/exchangers that are activated during this process and contribute to ischemic neuronal death. Glial cells that support and interact with neurons are also affected by the activity of exchangers. Endothelial cell channels/exchangers play an important role in BBB disruption and brain edema formation. Thus, these proton-sensitive channels/exchangers contribute to ischemic brain injury via various cell types and mechanisms, providing novel therapeutic targets for stroke intervention.

Figure 2. Structure and ionic permeability of ASIC channels

A. Phylogeny tree illustrates the subfamily members of ASIC channels and their distribution in the central nervous system (CNS) and peripheral nervous system (PNS). B. Predicted structural topology of ASIC channel. ASIC channel has a large extracellular domain, two putative transmembrane spans and two short intracellular termini. Functional ASICs are trimers, which predominantly conduct Na⁺. ASIC1a is the only homomeric ASIC that is substantially permeable to Ca²⁺. ASIC2b and ASIC4 do not form functional proton-gated homomeric channels. For more details see review (Grunder and Chen, 2010).

Figure 3. Contributions of NMDAR, ASICs, and TRPM7 channels to intracellular Ca²⁺ overload and neuronal injury in cerebral ischemia

During cerebral ischemia, the loss of Na⁺-K⁺-ATPase activity due to the shortage of ATP causes depolarization of neurons. Increased depolarization triggers uncontrolled release of the excitatory neurotransmitter glutamate. Excessive release of glutamate over-stimulates NMDA receptors, causing Ca²⁺ overload and neuronal injury. In addition, deprivation of oxygen makes anaerobic glycolysis become the primary source for ATP production. Anaerobic glycolysis causes the buildup of lactic acid, resulting in tissue acidosis. Extracellular acidosis activates ASIC1a, causing Ca²⁺ overload and neuronal injury. Following brain ischemia, increased generation of ROS, reduced intracellular Mg²⁺/ATP content, and reduction of extracellular divalent cations all facilitate the activation of TRPM7 channels, contributing to Ca²⁺ and Zn²⁺ entry and deteriorating the ischemia outcome. In addition to activating ASIC1a, extracellular acidosis may potentiate the activation of TRPM7 channels and subsequent Ca²⁺/Zn²⁺ toxicity.

Figure 4. Structure and ionic permeability of TRPM channels

A. Phylogeny tree illustrates the subfamily members of TRPM channels, based on reference (Nilius and Owsianik, 2011). B. Predicted structural topology of TRPM7 channel. TRPM7 channel has six putative transmembrane spans (TM) and a cation-permeable pore region formed by the loop between TM5 and TM6. TRPM7 is permeable to Na⁺ and divalent cations including Ca²⁺ and Mg²⁺, and trace metal ions Zn²⁺. TRPM7 is a chanzyme with an atypical C-terminal α-kinase domain.

Figure 5. Structure and regulatory sites of the NHE1 protein

NHE1 is composed of 813–822 amino acids, with 12 transmembrane (TM) segments and cytoplasmic N- and C-terminal domains. The cytoplasmic domains that are important in regulation or protein-protein interaction, the positions of reentrant loops, and the membrane-associated segments are illustrated. The associated factors, phosphatidylinositol 4, 5-bisphosphate (PIP2), ERM protein family (ezrin, radixin and moesin), calmodulin (CaM), and calcineurin homologous protein (CHP) are also shown in their approximate known binding sites. “P” indicates the approximate site for phosphorylation of the cytosolic tail of the protein mediated by extracellular signal related-kinase (ERK1/2), p90 ribosomal S kinase (p90^RSK), Nck-interacting kinase (NIK), or p160 Rho-associated kinase (p160ROCK) (This figure was published in Luo and Sun, 2007. Copyright © 2013 Bentham Science Publishers).

Figure 6. The role of NHE1 in microglial activation and migration following cerebral ischemia

Following cerebral ischemia, NHE1 functions to maintain microglial pH_i homeostasis and to sustain NOX function and “respiratory burst” in microglia. In turn, activated microglia release proinflammatory substances and participate in ischemic neuroinflammation and cell death. On the other hand, NHE1 also regulates microglial migration. NHE1 interacts with ERM proteins and functions as an anchoring point for actin filaments, contributing to membrane protrusion and microglial movement. NHE1 activity also maintains an alkaline pH_i in lamellipodia, which facilitates the pH_i-sensitive actin binding proteins actin depolymerizing factor (ADF)/cofilin function during microglial movement. In addition, NHE1-mediated Na⁺ influx triggers the NCX_rev operation and [Ca²⁺]_i rise, which further facilities ERM activation and actin accumulation. Taken together, NHE1 is an important regulator in microglial function and microglial-mediated inflammatory responses.