Correspondence to: Cecilia M. Canessa, 333 Cedar Street, New Haven, CT 06520-8026. Fax:(203) 785-4951 E-mail:cecilia.canessa{at}yale.edu.
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Abstract |
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The acid-sensitive ion channels known as ASIC are gated by external protons. A set of these channels is expressed in dorsal root ganglion neurons where they may participate in the transduction of mechanical and nociceptive stimuli. Here, we have examined the single-channel properties of channels formed by the subunits ASIC2 and ASIC3 expressed in Xenopus oocytes using outside-out patches. The mean single-channel current-voltage relationship is linear with a slope conductance of 18 pS between -80 and -40 mV in 150 mM Na+ outside and 150 mM K+ inside the patch pipet. The selectivity for monovalent cations has the sequence Na+ > Li+ > K+. Divalent cations such as Ca2+ do not permeate, but instead block the channel when applied to the extracellular side. External protons increase the probability of channels being open to a maximum of 0.8 with an EC50 of 16 ± 4 µM and a Hill coefficient of 2.7 ± 0.3, whereas the mean single-channel current amplitude is independent of external pH. Analysis of the kinetics of single channels indicates the presence of at least four modes of activity (Mod1 to Mod4) in addition to an inactivated state. Three of the modes exhibit distinct kinetics, and can be unambiguously identified on the basis of open probability (PoMod1 = 0.5 ± 0.05; PoMod2 > 0.9 ± 0.05; PoMod3 < 0.1). Mode 4, which has a Po in the range of 0.50.8, may constitute a distinct mode or alternatively, it represents transitions between the other three modes of activity. Increasing [H+]o increases the frequency of entering the modes with high Po (modes 1, 2, and 4) and the time the channel spends in the modes with high activity.
Key Words: protons, pH, degenerins, nociception, mechanoreceptor
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INTRODUCTION |
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The acid-sensitive ion channels (ASIC)1 are a family of proteins closely related in amino acid sequence and structure to the epithelial sodium channel (ENaC) and to the degenerins from Caenorhabditis elegans (mec-4, mec-10, and deg-1). They constitute a class of ion channels known as the DEG/ENaC superfamily (
Characteristic of ASIC is that external protons ([H+]o) activate the channels (
The physiological roles and functional pathways that depend on ASIC have not been well defined. However, selective expression of some of these proteins in dorsal root ganglion (DRG) neurons has suggested that they may participate in nociception and/or mechanoperception (
On the other hand, the idea that ASIC may represent a mechanoreceptor arose from the homology of these channels with the degenerins. The degenerins were initially identified by screening animals for deficits in light touch (
Several lines of evidence suggest that these two proteins form heteromeric channels. When the two subunits are coexpressed in heterologous systems, such as the oocyte, they induce currents larger in magnitude and with different properties than if any of the two subunits is expressed alone. Biochemical association of ASIC2 and ASIC3 has been shown in vitro by coimmunoprecipitation of the two proteins tagged with six-His and FLAG epitopes in oocytes (
The purpose of this study is to describe the effects of external protons and the properties of unitary currents of channels formed by the association of ASIC2 and ASIC3 to understand their functional role in sensory neurons.
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MATERIALS AND METHODS |
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Expression of Channels in Xenopus Oocytes
Channels were expressed in stage V and VI Xenopus laevis oocytes injected with 2 ng of cRNA from each ASIC2 and ASIC3. cRNAs were synthesized with T7 RNA polymerase from linearized plasmids containing the full-length cDNAs of ASIC2 and ASIC3 cloned by reverse transcriptasePCR from rat mRNA. Oocytes were incubated at 19°C for 25 d before making recordings. Before patching, oocytes were placed in an hyperosmotic solution for several minutes, and the vitelline membrane was removed manually with fine forceps.
Single-channel Recording
Currents were recorded from ASIC2-3 channels using the outside-out configuration of the patch-clamp technique. Experiments were performed on patches containing single channels unless indicated in the text. We determined the number of channels in the patch by performing recordings of several minute durations and counting the number of transition levels observed in the patch. We also applied a solution of pHo 4.0 to the outside-out patch, which is expected to maximally activate the channels present in the patch. Patches containing multiple channels were discarded from kinetics analysis.
Pipets were pulled from borosilicate glass (LG16; Dagan Corporation) using a micropipet puller (model PP-83 Narishige; Scientific Instrument Lab), fire-polished to a final tip diameter of 1 µm, and coated with Sylgard. When pipets were filled with solutions, they had resistances of 1020 M. Single-channel currents were recorded with an Axopatch-200B amplifier (Axon Instruments) using a TL-1 interface and pClamp6.0 software both from Axon Instruments. The data were filtered at 25 kHz, digitized at a sampling interval of 40 µs, and stored on a computer hard disk for analysis. For display, data were filtered with a digital Gaussian filter to 1 kHz.
All experiments were carried out at room temperature. The composition of solutions is given in millimolars. Incubation solution for oocytes: 96 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES, adjusted to pH 7.4. Hyperosmotic solution to remove the vitelline membrane: 220 NMDG, 220 aspartic acid, 2 MgCl2, 10 EGTA, and 10 HEPES, adjusted to pH 7.4 with KOH. Solution in the recording microchamber (extracellular): 150 NaCl, 1.8 CaCl2, and 10 Mes-Tris, adjusted to pH 7.4. For activation of ASIC channels, outside-out patches were perfused with solutions of composition identical to the extracellular solution but buffered to a lower pH, either 6.0, 5.0, 4.0, or as indicated in the experiment. To examine the selectivity of the channel for various cations, we replaced 150 NaCl with 150 LiCl or 150 KCl in the perfusion solutions, which were buffered to pH 4.0 for maximal activation of channels. Pipet solution (intracellular): 150 KCl, 5 EDTA, and 5 HEPES, adjusted to pH 7.4 with KOH.
Application of Solutions of Low pH to Outside-out Patches
The starting solution facing the tip of the pipet corresponding to the bath solution was always pH 7.4. Channels were activated by applying solutions of low pHo to outside-out patches using a solution delivery device that achieves complete solution changes within 20 ms (model SF-77B perfusion fast-step; Warner Instrument Corp.). Typically, three solutions were continuously flowing from three squared glass tubes (ID 0.7 mm). The patch pipet was initially positioned in front of the middle tube, which delivered a solution of pHo 7.4, and then moved with a voltage-command step to either of the other tubes, which delivered test solutions of lower pH: 6.0, 5.0, or 4.0, or as indicated in the particular experiment.
Whole-cell Recordings
For two-microelectrode recordings, current and voltage electrodes were pulled from borosilicate glass and filled with 3 M KCl. The resistance was kept in the range of 0.51 M. Oocytes were clamped at -60 mV and current-voltage relations were generated by changing the membrane potential from -150 or -100 to 50 or 100 mV in 10-mV incremental steps of 200 ms duration. Currents were recorded with an oocyte clamp (model OC-725B; Warner Instrument Corp.), digitized with a sampling rate of 100 Hz (model ITC-16; HEKA), and the data were stored on the hard disk of a PC. The oocyte chamber (volume of 300 µl) was continuously perfused at 12 ml/min. The composition of the standard bath solution contained (in mM) 100 NaCl, 2 KCl, and 1 CaCl2; bath solution without Na+ contained (in mM) 50 CaCl2 and 2 KCl; and bath solution with Na+ and Ca2+ contained (in mM) 100 NaCl, 2 KCl, and 50 CaCl2. All solutions were buffered with 10 HEPES and 10 MES, and the pH was adjusted to 7.4 or 4.0. Sorbitol was added to keep osmolality at 250 mOsm.
Data Analysis
Amplitude histograms were binned into equally spaced bins and fitted with the sum of two Gaussian components, using the method of least-squares minimization. Lists of the duration of single-channel events were generated using the half-amplitude threshold criterion. Histograms of the distributions of the open and closed dwell times were plotted as the logarithm of the dwell-time duration, with a square root transformation of the ordinate, and fitted with the sum of several exponential components using the maximal likelihood method of least-squares minimization. Data points represent the mean of at least three independent measurements ±SD.
Definition of Modes of Activity.
Only patches containing single channels were examined. Visual inspection of these records revealed that the kinetics of ASIC2-3 channels are not stationary but exhibit modes of activity. This impression was confirmed by analysis of Po with stability plots. We selected, from 3 to 5 min of continuous recording from the same patch, segments with low Po (<0.1), intermediate Po (0.5), and high Po (>0.9). Selected segments from each of the three groups were analyzed together as a continuous record and reexamined for kinetics and Po. Misclassified segments were removed from further analysis. Data from the three groups (modes 1, 2, and 3) were used for generating histograms of open and closed events. The rest of the records, comprising 3040% of channel activity, were considered as a mixture of modes (mode 4) or as unclassified.
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RESULTS |
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Identification of Single ASIC2-3 Channels in Oocytes
Exposure of outside-out patches to solutions of low pHo evoked single-channel activity in membranes from oocytes injected with ASIC2-3 cRNAs but not in uninjected oocytes. On average, the frequency of detecting acid-activated channels in injected oocytes was 7 out of 10 patches, whereas control oocytes did not express acid-activated channels. Fig 1 shows representative examples of unitary currents elicited by perfusion with an external solution buffered at pHo 4.0. The spikes in the recording indicate the change of solutions. No channel activity is detected at pHo 7.4; when the pHo is changed to 4.0, a channel opens (downward deflections) and it closes after returning to pHo 7.4. When multichannel patches were exposed to several sequential changes of pHo, the number of activated channels progressively decreased. This effect was more pronounced at high [H+]o.
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Conductance and Selectivity of ASIC2-3 Channels
Fig 2 shows all-points amplitude histograms of currents evoked by pHo 6.0, 5.0, and 4.0 recorded with 150 mM Na+ in the external solution and 150 mM K+ in the pipet. At -80 mV of membrane potential, the amplitude of the unitary current is -1.8 ± 0.04 pA with solutions of pHo 6.0, 5.0, and 4.0. The shoulder between the peaks and the small differences in conductance at the various pHo are mainly due to short events that do not reach full amplitude.
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Current-voltage relationships from unitary currents were obtained at pHo 4.0 in the presence of 150 mM external Na+, Li+, or K+ and 150 mM internal K+ (Fig 3). The amplitude of the unitary currents is larger with Na+ than with Li+, and larger with Li+ than with K+ outside. Under these experimental conditions, the slope conductance calculated between -80 and -40 mV is 18 pS for Na+, 12 pS for Li+, and 11 pS for K+.
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In symmetric K+, the I-V curve is linear over the range of voltages from -100 to -20 mV, indicating that the unitary conductance of ASIC2-3 is not voltage dependent. With Li+ or Na+ outside and K+ inside, the I-V curves show slight inward rectification as expected from the higher conductance of these two ions over K+.
Effect of Ca2+ Ions on the Activity of ASIC2-3
From previous studies it has been reported that some of the ASIC channels are permeable to divalent cations; specifically, that ASIC1 conducts Ca2+ (
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To examine the possibility that Ca2+ blocks Na+ currents conducted by ASIC2-3, we compared whole-cell I-V relationships in the presence of 100 mM Na+ as the current carrier with and without 50 mM Ca2+ in the bath solution (Fig 4 C, squares and circles, respectively). As before, no currents are detected at pHo 7.4. The black symbols, representing currents at pHo 7.4 ± 50 mM Ca2+ are superimposed. pHo 4.0 induces large currents that are partially blocked by 50 mM Ca2+ (open symbols), 70% block at -100 mV. Other divalent cations such as Zn2+ and Mg2+ also block ASIC2-3 channels (data not shown). These experiments, together with the fact that we did not see activation of the very abundant endogenous Ca2+-activated Cl- channels from oocytes, indicate that ASIC2-3 is not permeable to Ca2+; on the contrary, divalent cations block this channel.
Properties of the Fast and Sustain Components of ASIC2-3 Currents
Whole-cell currents from cells expressing ASIC2-3 exhibit a biphasic behavior (
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The only significant and reproducible finding immediately after changing the pHo was on the number of active channels. Fig 5 B shows two such examples. Lowering the pHo activates two channels, one closes after the first two seconds, whereas the other remains open for the remainder of the recording. This was a frequent finding, and we used it routinely as a test to estimate the total number of channels in the patch. Another feature of the fast component is that it appears only when pHo is changed from 7.4 to a lower pHo, but not if the pHo is sequentially changed. For instance, if the patch pipet is exposed to 7.4 followed by 6.0, 5.0, and 4.0 without returning to pHo 7.4, the fast component is seen exclusively in the transition from 7.4 to 6.0 but not during the other pHo changes.
The amplitude of the unitary currents was identical in the initial and late phases (1.3 ± 0.2 pA) as shown by the all-points histogram in Fig 5 C. In addition, IV curves generated with various ions substitutions during the sustained phase showed that this component of the current has selectivity Na+ > K+ > Li+. Together, the results indicate that the fast and sustain components are produced by channels with the same conductance and selectivity. The difference seems to be on the kinetic state of the channels. The fast component is generated by a population of channels that soon after activation enter a long lastinginactivated state and remain inactive until the pHo returns to 7.4.
Relationship of Channel Activity and pHo
The activity of ASIC2-3 increases with increasing concentrations of external protons; the effect is seen within the range of pHo from 6.4 to 4.0. Lowering the pHo below 4.0 does not induce a further increase in activity.
Fig 6 shows representative traces of outside-out patches containing single channels activated by pHo 6.0, 5.0, and 4.0. At pHo 6.0, the open probability (Po) is low; in the 20-s recording shown in the top panel, the average Po is 0.03. Most of the channel openings are of short duration (downward deflections indicated by OS), and they occur mostly in clusters. The periods of activity are separated by closures of different duration, some of which last several seconds (CL). Stretches with few short openings where the Po approaches the zero value are common at pHo 6.0. At pHo 5.0, the channel is more active with an average Po of 0.25. The short openings appear with higher frequency and, in addition, openings of intermediate and long duration (OI and OL) are apparent. At pHo 4.0, there is a further increase in Po to 0.69 due to more frequent and longer openings.
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The number of apparent open and closed states was determined from the distributions of open and closed dwell times from patches containing single channels. To ascertain the number of channels in the patch, we first activated the maximal number of channels by exposing it to pHo 4.0. Patches that showed more than one current level at any time during the recording were discarded from kinetics analysis. Fig 7 shows the histograms of open and closed events at pHo 5.0. Three exponential components were required to describe the distribution of observed open events, suggesting that channels adopt at least three open states. The observed mean channel open times of the three distributions (mean time constant followed by relative area in parentheses) are as follows: 0.66 ms (0.74) for the short openings, 3.9 ms (0.21) for the intermediate openings, and 19 ms (0.05) for the long openings. Six exponential components were required to fit the distributions of the closed events. The mean time constant and relative area for each of the distributions are as follows: 0.55 ms (0.34), 3.3 ms (0.31), 14 ms (0.21), 75 ms (0.08), 606 ms (0.04), and 4,880 ms (0.02).
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DoseResponse Curve of Single-channel Currents to [H+]o
The percentage of time the channel spends in the open state increases with [H+]o. Fig 8 plots this relationship for data obtained from patches activated with increasing concentrations of [H+]o. For each pHo, the mean Po was calculated from at least 80,000 open and closed events. For some of the points in Fig 8, data from three to five patches were combined to have sufficient number of events for the analysis because in these experiments patches were perfused for only 10 s with each solution.
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We removed from the analysis the long quiescent periods (2,000 ms). This long closures were infrequent but were seen at all pHo. Its inclusion in the analysis would have introduced a large variability in the Po among patches activated by the same pHo because not all patches examined contained these events (1020 events out of 20,000). If present, the average Po would be markedly decreased.
The line in Fig 8 represents the fit of the data to the Hill equation: Po = PoMax/(EC50N/[H+]oN + 1), where PoMax is 0.8, [H+]o is the concentration of external protons in micromolars, EC50 is the apparent concentration of protons that produces 50% of the maximal activation, and N is the Hill coefficient. The values of the apparent EC50 and N obtained from the fitting were 16 ± 4 µM (pHo = 4.8 ± 0.25, and 2.7 ± 0.3, respectively.
Stability of Channel Kinetics and Modes of Activity
Inspection of single-channel records reveals that the kinetics are not stationary. Channels exhibit sudden changes in Po and in kinetics. To test for stability of the data and possible kinetic modes, we calculated the mean Po averaged over 5-s intervals and plotted it versus time. Fig 9 shows such plots for pHo 6.0, 5.0, and 4.0. As indicated before, Po increases with increasing [H+]o; however, channel activity fluctuates between periods with openings and periods of quiescence, a characteristic that was consistently seen at all pHo.
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Visual inspection of the records and correlation with the Po stability plots makes evident the presence of distinct modes of activity, which differ in kinetics and Po. By using Po as the criterion to classify modes of activity, at least three modes were readily recognized: mode 1 (Po < 0.10), mode 2 (Po = 0.5 ± 0.05), and mode 3 (Po > 0.9 ± 0.05; Fig 10). In mode 1, the channel oscillates rapidly between open and closed states producing a flicker appearance. Mode 2 exhibits high Po and contains long open events. In contrast, the open events in mode 3 are short, and they are interrupted by closed events of long duration given a low Po to this mode. Mode 4 (Po = 0.50.8) has less distinct properties, it may constitute a distinct mode or alternatively, it represents transitions of the channel between the other three previous modes.
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Single-channel patches were examined with the Po criterion to detect modes 1, 2, and 3. Data collected from several patches were used to generate the histograms shown in Fig 11. The distributions of open intervals are described by the sum of two exponentials, whereas the distribution of closed events required two, three, and four exponentials for modes 1, 2, and 3, respectively. Mode 1 contains predominantly intermediate openings, mode 2 contains all the long openings, and mode 3 contains almost exclusively the short openings. The values of the apparent time constants and the percentage of events corresponding to each component are given in the legend of Fig 11.
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Since the criterion for identifying the modes was Po, all of the long closed events appear in mode 3. These events represent the slowest component (apparent time constant of 5,209 ms) of the histogram of closed events from mode 3. However, the long closed state was not only associated with mode 3, but it could follow or precede any of the other modes, suggesting that this closed state is connected with more than one mode of activity.
The frequency of the activity modes is pHo dependent. At pHo 6.0, the channel spends 98 ± 5% of the time in mode 3 and in the long closed state. At pH 5.0, the channel enters all modes and remains: 59 ± 10% in mode 3, 9 ± 3% in mode 2, 7 ± 4% in mode 1, and 25 ± 5% in mode 4. At pHo 4.0 the channel also enters all modes but spends more time in the high Po modes: 25 ± 7% in mode 2, 15 ± 15% in mode 3, 10 ± 7% in mode 1, and 50 ± 10% in mode 4 or unclassified.
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DISCUSSION |
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Properties of the Unitary Currents
This is the first report that describes the properties of unitary currents of channels formed by association of ASIC2 and ASIC3 subunits. Our studies revealed that these channels have a slope conductance of 18 pS measured in solutions containing 150 mM Na+ outside and 150 mM K+ inside. The permeability for monovalent cations has the sequence Na+ > Li+ > K+. We did not detect permeability for Ca2+; on the contrary, millimolar concentrations of Ca2+ and other divalent cations block ASIC2-3. These results indicate that opening of ASIC2-3 in vivo produces depolarization of the membrane potential but not activation of Ca2+-mediated second messengers in DRG neurons.
In symmetric solutions, the I-V relationship of unitary currents is linear. Thus, ASIC2-3 is not voltage dependent, a result that agrees with previous observations done on whole-cell currents from injected Xenopus oocytes (
Activation by [H+]o
External protons activate ASIC2-3 in a concentration-dependent manner by increasing the Po. The relation between Po and [H+]o was described by a slope to the power of 2.7, suggesting that two or more H+ ions are required for channel opening. Since [H+]o increased the number of openings and also the mean channel open time, both cooperativity of H+ on channel opening and an effect on prolonging the open time, could contribute to the N greater than two.
Inspection of records containing single channels indicates that ASIC2-3 exhibits complex kinetics. Analysis of dwell-time histograms of open and closed events revealed the presence of at least three open and six closed states. In addition, channels exhibit modes of activity that can be classified, on the basis of Po and kinetics, into four modes; modes 13 are distinct, whereas mode 4 could represent sudden shifts between the other three apparent modes of activity.
ASIC2-3 also exhibits an inactivated state that forms the basis of the fast component of the macroscopic current. Inactivation occurs only in a population of channels and it takes place shortly after lowering the pHo. To reactivate these channels with H+o, the pHo must first return to 7.4.
The mechanism by which the channel enters the different conformations defining each of the modes of activity is unknown. Sudden changes in kinetic states have been observed on other channels such as the glutamate-activated channels (
Detection of different modes of activity can give information about the underlying molecular mechanism and allow more direct data analysis. A comprehensive model that describes the kinetics of ASIC2-3 should include the various modes of activity and the way they are connected. Although we have not included in this work a quantitative description of the kinetics of ASIC2-3, our more qualitative studies of the single-channel behavior provide a means to distinguish ASIC2-3 from other channels in DRG neurons.
Physiological Significance of Activation by Protons
The question remains whether H+ ions gate ASIC2-3 or if some other kind of stimulus opens this channel under physiological conditions. Many investigators have reported the presence of various proton-activated currents in DRG neurons (
The recent finding that knockout of the ASIC2 gene in mice reduces touch (
Although this work does not solve the above questions, the single-channel properties we have described here need to be consistent with the functional role(s) ultimately assigned to ASIC in the peripheral nervous system.
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Footnotes |
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1 Abbreviations used in this paper: ASIC, acid-sensitive ion channels; DRG, dorsal root ganglion; ENaC, epithelial sodium channel; Po, open probability.
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Acknowledgements |
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This work was supported by the National Institutes of Health grant K01HL56163 to C. Canessa. The work was done under the tenure of an American Heart Association Postdoctoral Fellowship to P. Zhang.
Submitted: 5 December 2000
Revised: 30 April 2001
Accepted: 1 May 2001
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