Intracellular acidification evoked by moderate extracellular acidosis attenuates transient receptor potential V1 (TRPV1) channel activity in rat dorsal root ganglion neurons (original) (raw)
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TRPV1 Acts as Proton Channel to Induce Acidification in Nociceptive Neurons
Journal of Biological Chemistry, 2004
1 The abbreviations used are: pH ext , extracellular pH; pH i , intracellular pH; TRPV1, transient receptor potential vanilloid type 1; DRG, dorsal root ganglia; CFP and YFP, cyan and yellow fluorescent proteins; BCECF/AM, 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester; HEK293, human embryonic kidney 293; FRET, fluorescence resonance energy transfer; [Ca 2ϩ ] i , intracellular Ca 2ϩ concen-
Frontiers in Cellular Neuroscience, 2020
Acid-sensing ion channels (ASICs) are H +-activated neuronal Na + channels. They are involved in fear behavior, learning, neurodegeneration after ischemic stroke and in pain sensation. ASIC activation has so far been studied only with fast pH changes, although the pH changes associated with many roles of ASICs are slow. It is currently not known whether slow pH changes can open ASICs at all. Here, we investigated to which extent slow pH changes can activate ASIC1a channels and induce action potential signaling. To this end, ASIC1a current amplitudes and charge transport in transfected Chinese hamster ovary cells, and ASIC-mediated action potential signaling in cultured cortical neurons were measured in response to defined pH ramps of 1-40 s duration from pH 7.4 to pH 6.6 or 6.0. A kinetic model of the ASIC1a current was developed and integrated into the Hodgkin-Huxley action potential model. Interestingly, whereas the ASIC1a current amplitude decreased with slower pH ramps, action potential firing was higher upon intermediate than fast acidification in cortical neurons. Indeed, fast pH changes (<4 s) induced short action potential bursts, while pH changes of intermediate speed (4-10 s) induced longer bursts. Slower pH changes (>10 s) did in many experiments not generate action potentials. Computer simulations corroborated these observations. We provide here the first description of ASIC function in response to defined slow pH changes. Our study shows that ASIC1a currents, and neuronal activity induced by ASIC1a currents, strongly depend on the speed of pH changes. Importantly, with pH changes that take >10 s to complete, ASIC1a activation is inefficient. Therefore, it is likely that currently unknown modulatory mechanisms allow ASIC activity in situations such as ischemia and inflammation.
Regulation of TRPV5 Single-Channel Activity by Intracellular pH
Journal of Membrane Biology, 2007
The transient receptor potential channel TRPV5 contributes to the apical entry pathway for transcellular calcium reabsorption in the kidney. Acid load causes hypercalciuria in animals and humans. We have previously reported that intracellular protons directly inhibit TRPV5. Here, we examined the effects of intracellular pH on single-channel activity of TRPV5. We found that TRPV5 channels exhibit full and subconductance open states in excised inside-out patches of Chinese hamster ovary cells. The slope conductance values (Na + as a charge carrier, between -25 and -75 mV) for full and subconductance opening at intracellular pH 7.4 were 59 ± 6 and 29 ± 3 pS, respectively. Intracellular acidification caused a small decrease in single-channel conductance. Importantly, intracellular acidification decreased open probability for the full and subconductance states and increased probability for closing. To investigate how intracellular protons decrease open probability of the channel, we proposed a simple three-state model for open-subconductance-closed state transition and examined the effects of acidification on the respective forward and reverse rate constants. We found that intracellular acidification decreases opening of TRPV5 predominantly by promoting a transition from the subconductance to the closed state. Thus, intracellular acidification directly inhibits TRPV5 by causing a conformational change(s) leading to a decrease of open probability of TRPV5 as well as of the single-channel conductance.
Low pH facilitates capsaicin responses in isolated sensory neurons of the rat
Neuroscience Letters, 1996
The effects of capsaicin (CAPS; 30 nM, 300 nM, 3/~M) and acidic solutions (pH 6.6, 6.1, 5.6, 5.1) were studied in dorsal root ganglion (DRG) neurons from adult rats in short term culture using the whole cell patch-clamp technique and a system for fast drug application. At -60 mV holding potential, both CAPS 30 nM and 300 nM for 10 s did not induce a significant membrane current in pH 7.3. The first response to 3/zM CAPS at pH 7.3 yielded an inward current of 898 _+ 517 pA and with pH 6.1 the sustained protoninduced current was 365 -+ 153 pA. A more than additive current increase was observed when both agents were applied together even at subthreshold concentrations of CAPS or protons. Similar results were obtained at positive holding potential. Facilitation was also observed when extracellular pH 6.1 solution was applied immediately after discontinuation of 3/zM CAPS application but not when CAPS followed the application of pH 6.1 solution (n = 8). The proton-induced current as well as the CAPS-pH response both increased with proton concentration and showed the same short relaxation time relative to the CAPS response. The facilitation saturated near pH 5.6, and was present in repeated trials when responses to CAPS were markedly decreased due to tachyphylaxis. It is suggested that protonation of CAPS gated ion channels increases their open probability or conductance and modulates their kinetics.
AJP: Cell Physiology, 2004
Extracellular acidification has been shown to generate action potentials (APs) in several types of neurons. In this study, we investigated the role of acid-sensing ion channels (ASICs) in acid-induced AP generation in brain neurons. ASICs are neuronal Na+ channels that belong to the epithelial Na+ channel/degenerin family and are transiently activated by a rapid drop in extracellular pH. We compared the pharmacological and biophysical properties of acid-induced AP generation with those of ASIC currents in cultured hippocampal neurons. Our results show that acid-induced AP generation in these neurons is essentially due to ASIC activation. We demonstrate for the first time that the probability of inducing APs correlates with current entry through ASICs. We also show that ASIC activation in combination with other excitatory stimuli can either facilitate AP generation or inhibit AP bursts, depending on the conditions. ASIC-mediated generation and modulation of APs can be induced by extr...
Proceedings of the …, 2002
Acid-sensitive ion channels (ASIC) are proton-gated ion channels expressed in neurons of the mammalian central and peripheral nervous systems. The functional role of these channels is still uncertain, but they have been proposed to constitute mechanoreceptors and/or nociceptors. We have raised specific antibodies for ASIC1, ASIC2, ASIC3, and ASIC4 to examine the distribution of these proteins in neurons from dorsal root ganglia (DRG) and to determine their subcellular localization. Western blot analysis demonstrates that all four ASIC proteins are expressed in DRG and sciatic nerve. Immunohistochemical experiments and functional measurements of unitary currents from the ASICs with the patch-clamp technique indicate that ASIC1 localizes to the plasma membrane of small-, medium-, and large-diameter cells, whereas ASIC2 and ASIC3 are preferentially in medium to large cells. Neurons coexpressing ASIC2 and ASIC3 form predominantly heteromeric ASIC2-3 channels. Two spliced forms, ASIC2a and ASIC2b, colocalize in the same population of DRG neurons. Within cells, the ASICs are present mainly on the plasma membrane of the soma and cellular processes. Functional studies indicate that the pH sensitivity for inactivation of ASIC1 is much higher than the one for activation; hence, increases in proton concentration will inactivate the channel. These functional properties and localization in DRG have profound implications for the putative functional roles of ASICs in the nervous system.
Store-operated Ca2+-channels are sensitive to changes in extracellular pH
Biochemical and Biophysical Research Communications, 2005
The sensitivity of store-operated Ca 2+ -entry to changes in the extra-and intracellular pH (pH o and pH i , respectively) was investigated in SH-SY5Y human neuroblastoma cells. The intracellular Ca 2+ -stores were depleted either with 1 mM carbachol (CCH) or with 2 lM thapsigargin (TG). Extracellular acidification suppressed both the CCH-and TG-mediated Ca 2+ -entry while external alkalinization augmented both the CCH-and the TG-induced Ca 2+ -influx. Mn 2+ -quenching experiments revealed that the rates of Ca 2+ -entry at the thapsigargin-or carbachol-induced plateau were both accelerated at pH o 8.2 and slowed down at pH o 6.8 with respect to the control at pH o 7.4. Alteration of pH o between 6.8 and 8.2 did not have any significant prompt effect on pH i and changes in pH i left the CCH-induced Ca 2+ -entry unaffected. These findings demonstrate that physiologically relevant changes in pH o affect the store-operated Ca 2+ -entry in SH-SY5Y cells and suggest that endogenous pH o shifts may regulate cell activity in situ via modulating the store-operated Ca 2+ -entry.
Journal of Biological Chemistry, 2005
Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular protons. The ASIC3 subunit is largely expressed in the peripheral nervous system, where it contributes to pain perception and to some aspects of mechanosensation. We report here a PDZ-dependent and protein kinase C-modulated association between ASIC3 and the Na ؉ /H ؉ exchanger regulatory factor-1 (NHERF-1) adaptor protein. We show that NHERF-1 and ASIC3 are co-expressed in dorsal root ganglion neurons. NHERF-1 enhances the ASIC3 peak current in heterologous cells, including F-11 dorsal root ganglion cells, by increasing the amount of channel at the plasma membrane. Perhaps more importantly, we show that the plateau current of ASIC3 can be dramatically increased (10-30fold) by association with NHERF-1, leading to a significant sustained current at pH 6.6. In the presence of NHERF-1, the ASIC3 subcellular localization is modified, and the channel co-localizes with ezrin, a member of the ezrin-radixin-moesin family of actinbinding proteins, providing the first direct link between ASIC3 and the cortical cytoskeleton. Given the importance of the ASIC3 sustained current in nociceptor excitability, it is likely that NHERF-1 participates in channel functions associated with nociception and mechanosensation. Physiopathological conditions such as ischemia, inflammation, tumors, or injury are associated with a decrease in extracellular pH, and tissue acidosis has been linked to pain in human volunteers (1-3). Nociceptive neurons display voltage-independent H ϩ-gated cationic currents, which are in part mediated by acid-sensing ion channels (ASICs) 2 (4, 5). The ASIC family comprises six isoforms encoded by four different genes and expressed in sensory and/or central neurons (6, 7). Functional ASICs are homomeric or heteromeric channels, probably tetramers, with different kinetics, external pH sensitivities, and tissue distribution