Regulation Of Acid-sensitive Ion Channels In The Central Nervous System

Acid-sensing ion channels (ASICs) are amiloride-sensitive and voltage-insensitive cation channels that are activated by extracellular H⁺. Four genes encoding six ASIC subunits, ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3 and ASIC4, have been cloned and demonstrated to be broadly expressed throughout the body including the central and peripheral nervous system. Increasing evidence has highlighted the role of ASICs in the central nervous system in various physiological and pathological processes such as learning and memory, emotion, retina function, ischemic brain injury and brain tumor. In addition, the contribution of ASICs to synaptic function has been recently elucidated.It is well known that ASICs are functionally regulated by some exo- and endogenous agents such as metal ions including physiologically relevant Ca²⁺, Zn²⁺, Mg²⁺ and toxically relevant Ni²⁺, Cd²⁺, Gd²⁺. Moreover, unraveling the interaction between metal ions and ASICs will provide the structure information, which is helpful to the design of novel drugs that specifically target these channels. Therefore, in our study, we examined the effects of Cu²⁺, an essential element, and Pb²⁺, a well-known neurotoxicant, on ASICs in the central neurons using whole-cell patch clamp combined with cell transfection and Ca²⁺ imaging. Besides, using whole-cell patch clamp, we made preliminary investigation on the ASICs in the hypothalamus, the region of the brain that controls a large number of bodily functions. The major findings are summarized as follows:1. The modulation of ASICs by Pb²⁺At a holding potential of -50 mV, extracellular Pb²⁺ rapidly and reversibly inhibited the amplitude of ASIC currents in the acutely dissociated hippocampal CA1 and spinal dorsal horn (SDH) neurons of the rat in a concentration-dependent manner. The results from the CHO cells transfected with five major ASIC subunits: 1a,1b, 2a, 2b and 3 in combination showed that the ASIC currents mediated by ASIC1a, 1b, and 3 were inhibited by Pb²⁺. Since ASIC1b and 3 have been reported to be expressed in the peripheral nervous system of the rat, we suggest that ASIC1a is the main molecular target of ASICs in the central nervous system. Next, we studied the mechanisms underlying the modulation of ASICs by Pb²⁺ in the SDH neurons in which ASIC currents are mainly mediated by ASIC1a. We found that Pb²⁺-induced inhibition of ASIC currents was voltage-independent, and not modified by intracellular signaling messengers. These results suggest that the action site(s) of Pb²⁺ are located on entracellular domain of these channels. The non-competitive interaction of Pb²⁺ and amiloride with ASIC currents also supports this speculation.Furthermore, we found that Pb²⁺ shifted activation curve of ASICs in a parallel manner without significantly altering the maximal value and Hill coefficient, suggesting that Pb²⁺ reduced the affinity of ASICs for H⁺ in a competitive manner. Notably, the Ca²⁺ modulation of ASICs is also pH-dependent manner. To examine their possible interactions in inhibiting these channels, we co-applied the two divalent cations to ASICs, and found that they modulated ASICs in a distinct fashion. Paukert et al. has ever proposed a model to explain the mechanism underlying the modulation of ASIC1a by Ca²⁺. In this model, Ca²⁺ could bind to two action sites located on ASIC1a, a blocking site and a putative modulating site, to modulate ASIC1a currents. We modified and employed this model to explain the modulation of ASICs by Pb²⁺. When binding to the blocking site, Pb²⁺ blocked ASIC currents in the open state of the channel, whereas binding to the modulating site, Pb²⁺ induced a conformational change of the channel protein, resulting in an inhibition of ASIC currents. When binding to either blocking site or modulating site, Pb²⁺ influences the effects on ASICs by H⁺.2. The modulation of ASICs by Cu²⁺Cu²⁺ is the essential element in human body and plays important roles in maintaining normal functions of the brain. Abnormal amount of Cu²⁺ are associated with some neurological diseases. In the cultured hypothalamic neurons, we found that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Similar results were obtained from hippocampal and cortical neurons of the rat. These results identified Cu²⁺ as an endogenous modulator of ASIC activity. Further study showed that the Cu²⁺ modulation of ASICs was independent of change in membrane potentials suggesting that the action site(s) of Cu²⁺ on ASIC channel is located on extracellular domain. In addition, the effects of Cu²⁺ on activation curve of ASICs indicate a non-competitive mechanism, which implies an allosteric modulation. The prolonged desensitization of ASICs by Cu²⁺ supports this speculation. Employed three different modes of drug application, we found that the inhibitory effect in pre-treatment protocol was statistically identical to that of the liner summation from co-application protocol and sequential application protocol. Based on these results, we speculate that two separate action sites, a blocking site and a modulating site, are located on the extracellular domain of ASICs. Cu²⁺ reduced sensitivity of ASICs to H⁺ via these action sites, which accounts for the reduced amplitude of ASIC currents, and the prolonged desensitization of these channels. Since the two sites are separate, we thus observed a cumulative inhibition caused by Cu²⁺ binding to both sites.Another finding in this study was that Cu²⁺ reversibly attenuated the increased membrane excitability mediated by activation of ASICs. This negative modulation may be helpful to maintain normal neuronal excitation during synaptic transmission. From the results from CHO cells expressing ASIC1a subunit, we found this subunit is inhibited by Cu²⁺. Since ASIC la-mediated acidotoxicity is the important mechanism of ischemic brain injury, Cu²⁺-induced inhibition of ASIC1a may be protective in the conditions of stroke.3. The ASICs in the hypothalamusWe recorded proton-induced cation currents in the cultured hypothalamic neurons of the rat. These transient inward currents with an activation threshold of around pH 6.8, were mainly carried by Na⁺, and reversibly blocked by amiloride, the known ASIC antagonist. Based on these typical electrophysiological and pharmacological properties, we attribute these currents in the hypothalamic neurons to ASICs. Further study exhibited that homomeric ASIC1a channels and heteromeric ASIC1a+2a channels may mediate the ASIC currents in the hypothalamic neurons. We can not exclude the presence of heteromeric ASIC1a+2b responsible for ASIC currents. A novel finding in this study is that functional ASIC3 channels might be present in the central neurons of the rat. These conclusions have been supported by the preliminary results from RT-PCR. Another finding in this study is that membrane depolarization in the hypothalamic neurons could be induced by a mild and moderate drop in extracellular pH that is within the range locally reached by pH fluctuations during normal synaptic transmission or due to tissue acidosis occurring in the pathological conditions. Therefore, it is conceivable that the hypothalamic ASICs may regulate hormone release from hypothalamic neurons via ASIC-triggered membrane depolarization. Considering that different ASIC subtypes are involved in many physiological and pathological processes, and the hypothalamus mediates distinct functions, the presence of multiple ASIC subunits suggests their potential roles in this important brain region.