Neuroscience Research 24 (1996) 109-116
of neuronal function by intracellular pH
Kyoh-Ichi Takahashi*“, David R. Copenhagenb ‘Division bDepartments
of Commercial Sciences, Hiroshima Shudo University, I71 7 O&&a, Numata-cho, Asaminami-ku, Hiroshima 731-31, Japan and Physiology, University of California San Francisco, San Francisco, CA 94143-0730, USA
Received 23 October 1995; accepted 8 November 1995
Abstract Recentstudieshave revealedthat excitation of specificnervepathwayscan producelocalizedchangesof pH in nervoustissue. It is importantto determineboth how thesepH changesare generatedand,evenmoreimportantly, how the excitability of neurons in the localizedareasare affected.Evidenceindicatesthat activation of both y-aminobutyric acid (GABA) and t-glutamatereceptor channelsin inhibitory and excitatory pathways,respectively,can raiseextracellularpH (pH,) andlower intracellularpH (pHi). At the target location, it hasbeenshownthat severaltypes of voltage-gatedion channelsin neuronswere modifiedby a change in pHi. These studies,taken together, enable us to hypothesize that intracellular hydrogen ions (H’) might function as neuromodulatoryfactors,like other typesof intracellularsecondmessengers. This hypothesiswastestedby usinghorizontal cells enzymaticallydissociatedfrom catfish retina. We found that the high-voltage-activated(HVA) Ca’+ current, inward rectifier K+ current and hemi-gapjunctional current are modulatedby a changein intracellularH+ concentration,and that L-glutamatesup presses the HVA Ca’+ current by raisingthe intracellularH+ concentration.Theseobservationssupportthe hypothesisthat intracellular H+, acting as a secondmessenger, governsneuronalexcitability via modulation of ionic channelactivity. This article reviewsrecentstudiesof ours and otherson the effect of pHi upon neuronalfunction. Keywords:
Vertebrate retina; Horizontal cell; Neuronal function; Intracellular pH; Voltage-gatedion channel; L-Glutamate;
It has been commonly assumed that both extracellular pH @HO)of tissue and intracellular pH @Hi) in various excitable cells such as neurons and muscle fibers and in other cell types including oocytes are well regulated by homeostatic mechanisms. These mechanisms include transporter functions of the blood-brain barrier and the choroid plexus that maintain pH, and the transporters of the plasma membranes of cells that regulate pHi. Except for a few groups such as R.C. Thomas and his colleagues, little attention had been paid to changes of pHi * Corresponding author, Tel.: +81 82 830 1242; fax: +81 82 830 1326; e-mail: takahasiQshudo-u.ac.jp.
in excitable cells. In the last two decades, however, several techniques for measuring pHi have evolved to study how pHi is regulated, e.g., pH-sensitive electrodes (Boron and DeWeer, 1976; Roos and Boron, 1981; Thomas, 1984), pH-sensitive indicator dyes (Rink et al., 1982; Paradiso et al., 1987a,b). As a result, accumulating evidence suggests that pHi can change significantly but also under not only pathophysiological physiological circumstances, and that changes in pHi exert profound effects on the physiological and biophysical properties of cells. These discoveries argue for the case that intracellular hydrogen ions (H+) might be acting as second messengers for regulating the excitability of cells. In this article, recent advances in the study of pHi in neurons will be reviewed.
0168-0102/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-0102(95)00989-7
2. Regulatory mechanisms of intracellular
D. R. Copenhagen
As fundamental evidence for the regulation of pHi, one can consider the electromotive force of H+ across a nerve cell membrane. If H+ were passively distributed across the plasma membrane of neurons, the concentration of intracellular H+ could be predicted using the Nernst equation. For a typical membrane potential of -60 mV and for pH, of 7.4, a pHi of 6.4 would be expected if H+ were in electrochemical equilibrium across the membrane. However, typically net pHi is much higher in cells under these conditions. This implies that cells have mechanisms for H+ extrusion that maintain pHi at a higher level, and/or that cytosol has a strong buffering capacity for H+. Several types of exchangers in neurons have been identified that can produce a net extrusion of H+ (e.g., see the review article of Kaila, 1994). Three of these are well characterized: Na+/H+ exchangers, Cl-/HCO,exchangers and Na+-dependent Cl-/HC03exchangers (Moody, 1984; Chesler, 1990). The cytosolic constituents act as strong pH buffers. The cytosolic buffering power is defined as the amount of H+ (in mmol/l) that needs to be added to a solution to produce a 1 unit change in pH. Buffering power has been measured in a variety of cells using several methods. The buffering power in various types of neurons range from 10 to 50 mrnol/l per pH unit (Chesler, 1990). 3. Modulation
pH by neurotransmitters
Contrary to the notion of homeostatic mechanisms keeping pHi and pH, constant, recent studies have demonstrated that neuronal activity gives rise to changes in pH, as well as pHi (Chesler, 1990; Chesler and Kaila, 1992). Several studies have been carried out to elucidate the mechanism(s) underlying changes in pH, and pHi. Kaila and Voipio (1987) reported that y-aminobutyric acid (GABA) acidifies the dactyl opener muscle cells of the first walking leg of the crayfish by an eMux of bicarbonate ion (HCOj-) through anion-selective GABAA receptor channels. Later they observed the same phenomenon in rat hippocampal pyramidal neurons (Pastemack et al., 1993). Endres et al. (1986) reported that kainate, quisqualate and N-methyl-D-aspartate (NMDA), ionotropic Lglutamate receptor agonists, acidify motoneurons of the isolated spinal cord in a concentration-dependent manner, although the mechanism(s) remained unknown. Recently Paalasmaa et al. (1994) suggested that a Ca2+/H+ ATPase exists in plasma membrane of rat hippocampal neurons. When the intracellular Ca2+ concentration is increased by an influx of Ca2+ through L-glutamate receptor channels, the subsequent activation of the Ca”+/H+ ATPase acidifies the neurons.
Dixon et al. (1993) also found that in catfish retinal horizontal cells (Fig. 1A) L-glutamate induced an acidification (Fig. lB, 0.3 pH unit from the resting pHJ. The acidification was partially suppressed when superfused with a high concentration of kynurenic acid (5 mM) (unpublished data), suggesting either a direct influx of H+ through L-glutamate receptor channels per se or an influx of H+ evoked by an action of Ca’+/H+ ATPase which was activated by an increase of Ca*+ entering through L-glutamate receptor channels (Paalasmaa et al., 1994). The conclusions drawn from the results of these studies are that, even with homeostatic mechanisms to control pH, these inhibitory and excitatory neurotransmitters caused localized changes in pH, and pHi. 4. Modulation of voltage-gated ion channels by intracellular H+
Many studies have revealed that second messengers such as Ca2+, cyclic nucleotides and G-proteins modulate voltage-gated ion channels and ligand-gated receptor channels. Changes in pHi have also been known to modify several types of voltage-gated ion channels: Ca*+ currents (Umbach, 1982; Irisawa and Sato, 1986; Kaibara and Kameyama, 1988; Katzka and Morad, 1989; Mironov and Lux, 1991; Takahashi et al., 1993), inward rectifier K+ currents (Blatz, 1980; Moody and Hagiwara, 1982; Ito et al., 1992; Takahashi and Copenhagen, 1995), delayed rectifier K+ currents (Wanke et al., 1979), Na+ currents (Brodwick and Eaton, 1978). 5. An example of modulation of a calcium current by Lglutamate-inducedacidificaton
Catfish retinal horizontal cells express high-voltageactivated (HVA) Ca2+ channels (L-type), which are activated at around -40 mV and show no inactivation (Takahashi et a1.,1993). Fig. 2 shows the HVA Ca2+ current in a horizontal cell voltage-clamped with patch pipettes recorded in the whole-cell configuration. Fig. 2A shows the currents recorded as the clamp potential was stepped to progressively more depolarized levels from the holding potential of -50 mV. The records in Fig. 2B show the current evoked in the presence of 20 PM cadmium. Fig. 2C shows the cadmium-sensitive current obtained by subtraction (traces in Fig. 2B minus those in Fig. 2C). The current was also suppressed by (data not shown). The 100 PM nifedipine current-voltage (I-V) relationship is shown in Fig. 2D. Taken together, it is clear that the Ca*+ current recorded in horizontal cells is carried via the HVA Ca2+ channels. The amplitude of the HVA Ca2+ current in the horizontal cells was modified by a change of pHi. In order to change pHi, a NH&l prepulse technique was
B Glutamate (25
(50 pm ..:.:.:.:.:.:.> .. .. . .. -.*.*...*.*.*.. *.*.*.*.*.-.-.* . .. . .. ..
(100 pw .:.:.:.:.> :.:.>:.:.: .*,..*.*... .. . . . :.:.>:.:.: .*.*.-.-... . .. . .
Fig. I. Intracellular acidification of a horizontal cell evoked by L-glutamate. A: photomicrograph of a horizontal cell (*) enzymatically dissociated from a retina of cattish (Icutalurus punctatus) and kept at 14°C for 2 days in a culture medium supplemented with ~-15. B: a cell was incubated with 5 FM BCECF-AM for 5 mitt and then rinsed in the control supcrfusate for 40 min to allow the fluorescent signal to reach equilibrium. pHi was measured as the ratio of 440 nm:490 nm-induced fluorescence at 535 nm. L-glutamate caused the cell to acidify in a concentration-dependent manner. Reproduced from Dixon et al. (1993) by copyright permission of Cell Press.
# 5 0 I
300 msec -500
Fig. 2. HVA Ca2+ currents in a horizontal cell dissociated from cattish retina. Patch pipettes containing 130 mM Cs-gluconate were used to suppress several types of K+ currents expressed in horizontal cells. A: the horizontal cell was voltage-clamped at -50 mV in the whole-cell patchclamp mode. Pulses of I-s duration were applied to the cell in increments of 10 mV between -40 and +50 mV. 9: the same protocols as in A were employed in the presence of 20 CM cadmium. C: the responses in B were subtracted from those in A to eliminate leak currents, and revealed the HVA Ca2+ currents. D: the current-voltage relationship for this cell, measured 500 ms into the pulse, was obtained from data in C. Not all steps are shown in A-C for clarity. The currents were recorded in IS mM barium containing superfusate. Reproduced from Takahashi et al. (1993) by copyright permission of The Rockefeller University Press.
employed. The technique and results are described as follows. 5. I. Experiments designedto changepHi without changes in pH,
In order to study the electrophysiological effects of changes in pHi, the following techniques are commonly used. (1) The NH&l prepulse technique: a short exposure of the cell to NH&l causes an intracellular alkalinization and then an acidification following washout of NH&l. During the first moments of exposure to NH&I, NH3 quickly and NH4+ slowly enter the cell passively. Entering NH3 combines with intracellular H+ to produce NHd+, thereby alkalinizing the cell. As NH,+ enters it dissociates to form NH3 and H+,
thereby gradually reducing the initial rapid alkalinization of the cell. Following the washout of NH&l, NH3 leaves the cell rapidly, which leads to further dissociation of NH,,+ to NH3 and liberation of H+. This produces a rebound acidification that goes below resting Ph. (2) Exposure of the cell to weak acid: weak acids such as acetate and CO2 cause an intracellular acidification during exposure. Uncharged acids can penetrate cell membranes easily, after which they dissociate intracellularly to liberate H+, resulting in acidification. We employed primarily the NH&l prepulse technique, because the technique can be used to reliably change pHi of horizontal cells and to both alkalinize and acidify these cells (Takahashi and Copenhagen, 1992, 1995; Dixon et al., 1993, 1996; Takahashi et al., 1993). Fig. 3 shows a typical example. The cell was in-
D. R. Copenhagen
Fig 3. Modulation of pHi by NH&I (20 mM). A cell that was dissociated enzymatically from catfish retina was incubated with 5 PM BCECF-AM for 5 min, and then rinsed in the control superfusate for 40 min to allow the fluorescent signal to reach equilibrium. This cell had a slender axon with a bulbous terminal. BCECF was loaded in the cell body (triangular shape) and the terminal (round shape above the cell body). pHi was measured as the ratio of 440 nm:490 nm-induced fluorescence at 535 nm and displayed with pseudo-color before, during and after an application of NH&I. A: a 4.3 min exposure to NH&l resulted in alkalinization (b) from the resting pHi (a), followed by acidification (c) after the washout of NH&I. pHi recovered in I3 min (d). Note that changes in pHi were detected in not only the cell body but also the terminal. B: pHi was recorded every 5 s and was measured in the area surrounded by the dashed line in A. The pseudo-color images in A were obtained at the timings indicated in B.
cubated with 5 PM BCECF-AM (2’,7’-b&(2carboxyethyl)-5-(and 6) carboxyfluorescein, acetoxymethyl ester) for 5 min and then rinsed in the control superfusate for 40 mm to allow the fluorescent signal to reach equilibrium. In Fig. 3A, pHi as the ratio of 440 mu:490 nm-induced fluorescence at 535 nm was measured and displayed with pseudo-color, in which a high ratio indicates alkalinization. The ratio increased upon application of 20 mM NH&l, and decreased upon washout of NH&l. Fig. 3B shows the time course of change in pHi in control saline, in 20 mM NH&l-
containing saline and washout of NH&I. Fig. 33 shows that pHi shifted towards alkalinization upon application of 20 mM NH&l (see Fig. 3Ab) and then the washout led to acidification (see Fig. 3Ac). pHi recovered the original level 13 min after the washout. 5.2. Modulation of a calcium current by L-glutamateinducedacidification
Dixon et al. (1993) found that in cattish retinal horizontal cells L-glutamate suppressed the HVA Ca’+ cur-
D. R. Copenhagen
rent through intracellular acidification. In the present study, the effect of L-glutamate on HVA Ca*+ current and pHi were simultaneously examined. Fig. 4 shows an example of the change in pHi induced by 10 PM Lglutamate and by an application and washout of 20 mM NH&l. Fig. 4A shows that 10 PM L-glutamate acidilied the cell by 0.15 pH units. Subsequent exposure to 20 mM NH&l caused a 0.35 pH unit alkalinization, followed by an acidification that peaked 0.4 pH units below the resting pHi of 7.4. I-V relationships were investigated with a voltage ramp from -90 mV to +50 mV in control superfusate and during the times when pHi
was modified by L-glutamate or by NH&l. Fig. 4B shows I-V relationships of a HVA Ca*+ current measured before acidification, during the peak acidification, and after recovery from 10 pM L-glutamate exposure. Fig. 4C shows I-V relationships measured before, during, and at two subsequent times after washout of NH&l. The peak HVA Ca*+ current was enhanced in the. presence of NH&l, while the current was reduced during washout of NH&l. After recovery of pHi, the HVA current returned to the control level. When the cells were recorded with pipettes containing a high concentration of pH buffer to ‘clamp’ pHi, the effects of NH&l and L-glutamate were dramatically reduced (data not shown). From these observations and previously published works, we are led to two important concepts. The first concept is that L-glutamate, the transmitter released from photoreceptors, has two separate functions. It serves as a conventional excitatory neurotransmitter, as has been recognized for many years, but it also acts as a neuromodulator on the same postsynaptic neuron. By adjusting the magnitude of the Ca*+ currents, Lglutamate, on a longer time course, can adjust Ca*+dependent cellular functions. The second concept is that changes in intracellular H+ concentrations can modify a voltage-gated ion channel. Our finding (Dixon et al., 1993) showing that high intracellular pH buffering eliminates the effect of L-glutamate on the Ca*+ currents, demonstrates that H+ is an essential element in the pathway between L-glutamate receptor activation and Ca*+ current modulation. In the strictest sense, therefore, H+ must be considered a second messenger. Future studies will be required to establish whether H+ acts directly or indirectly on the Ca*+ channels and whether Ca*+ itself is part of the pathway. Preliminary results (Dixon et al., 1993) suggest that Ca*+ itself may
Fig. 4. Simultaneous recording of HVA Ca*+ current and pHi in a horizontal cell dissociated from cattish retina. The cell was recorded with a BCECF-filled pipette in the whole-cell patch-clamp mode. pHi was recorded continuously and voltage ramps were applied to the cell before, during, and after application of 10 CM L-glutamate or 20 mM NH&l. A: a 1 min exposure to L-glutamate resulted in a small reduction (0. I5 pH units, q from the resting pHi [Il. pHi recovered in 2.5 min [ZI. Subsequent application of NH&I produced a 0.35 pH unit alkalinization [II from the resting pHi q followed by a 0.4 pH unit acidification during the washout of NH&I q . pHi recovered 30 min after the washout of NH&I [il. B: the peak HVA Ca** current q was reduced approximately 25% at the peak of L-glutamateinduced intracellular acidification. Both the pHi and the HVA Ca*+ current recovered in 2.5 min [IL C: HVA Ca*+ current during an application of NH&I was increased 12% at the maximum alkalinization q and decreased 55% during the peak acidification following the washout of NH&I. Both the pHi and the HVA Ca*+ current recovered 30 min after the washout of NH&I q . Reproduced from Dixon et al. (1993) by copyright permission of Cell Press.
play only a minor role because the effects of Lglutamate on the Ca2+ current were readily apparent when the cells were studied with 10 mM EGTA in the recording pipette, a concentration which should buffer Ca2+ changes. 6. Different actions of intracellular
pH in a single cell
The inward rectifier K+ current (Takahashi and Copenhagen, 1995) as well as the hem&gap junctional current (Dixon et al., 1996) in cattish retinal horizontal cells were also modified via a change in pHi, like the HVA Ca2+ current (Takahashi et al., 1993). However the sensitivity to pHi of the inward rectifier and hemigap junctional currents differed from that of the HVA Ca2+ current. The HVA Ca2+ current was much more sensitive to intracellular acidification than to intracellular alkalinization, while the inward rectifier current was not changed by acidification below the resting pHi of 7.4 but was enhanced by alkalinization above 7.4. The hem&gap junctional current was very sensitive to both directions of changes in pHi. The physiological significance of how intracellular H+ concentration regulates the activities of these different channels requires further study. 7. Site(s) of modification
If future research establishes that H+ directly acts on the Ca2+ channel protein and that there are no other second messengers in the cascade or other intermediate steps, we can suggest that a candidate site for the action of H+ would be on histidine residues. Histidine is the only amino acid with a titratable site near physiological pH (6.0). The pK for this action (the pK of the imidazole ring of the free amino acid histidine) seems too low to account for our data (Takahashi et al., 1993; Dixon et al., 1993,1996; Takahashi and Copenhagen, 1995), however the actual titration point can be influenced by the molecular environment near the histidine residue. Therefore, it seems reasonable that histidine groups on the internal surface of the channel proteins may provide functional sites for modulating channel activity. 8. Closing remarks L-glutamate and GABA are major neurotransmitters in the central nervous system. In this article we reviewed examples in which the transmitters can evoke not only electrical signals but also changes in pHi through activation of postsynaptic receptors, and that the pHi changes can modulate voltage-gated ion channel activity. Because the changes in pHi are also thought to be large enough to influence enzymatic activity, intracellular H+ in neurons appears likely to play other crucial roles in maintaining normal neuronal function.
Acknowledgments A part of the original work reported here was performed in collaboration with Drs. Dixon and Kaneko. The original works reported here were supported by a Grant in Aid for Scientific Research from the Ministry of Education, Science and Culture, by a grant from the Ichiro Kanehara Foundation, by a grant from the Japanese Society for Promotion of Science to K.I.T., and by grants from NIH and NSF to D.R.C. References Blatz, A.L. (1980) Chemical modifiers and low internal pH block inward rectifier K channels. Fed. Proc., 39: 2073 Boron, W.F. and DeWeer, P. (1976) Intracellular pH transients in squid giant axons caused by COZ, NH,, and metabolic inhibitors. J. Gen. Physiol., 67: 91-112. B&wick, MS. and Eaton D.C. (1978) Sodium channel inactivation in squid axon is removed by high internal pH or tyrosine-specific reagents. Science, 200: l494- 1496. Chesler, M. (1990) The regulation and modulation of pH in the nervous system. Prog. Neurobiol., 34: 401-422. Chesler, M. and Kaila, K. (1992) Modulation of pH by neuronal activity. Trends Neurosci., 15: 396-402. Dixon, D.B., Takahashi, K.-I. and Copenhagen, D.R. (1993) Lglutamate suppresses HVA calcium current in cattish horizontal cells by raising intracellular proton concentration. Neuron, 11: 267-277. Dixon, D.B., Takahashi, K.-I., Bieda, M. and Copenhagen, D.R. (1996) Quinine, intracellular pH and modulation of hemi-gap junctions in catfish horizontal cells. Vision Res., in press. Endres, W., Ballanyi, K., Serve, G. and Grafe, P. (1986) Excitatory amino acids and intracellular pH in motoneurons of the isolated frog spinal cord. Neurosci. Lett., 72: 54-58. Irisawa, H. and Sato, R. (1986) Intra- and extracellular actions of proton on the calcium current of isolated guinea pig ventricular cells. Circ. Res., 59: 348-355. Ito, H., Vereecke, J. and Carmeleit, E. (1992) Intracellular protons inhibit inward rectifier KC channel of guinea-pig ventricular cells. Plliigers Arch., 422: 280-286. Kaibara, M. and Kameyama, M. (1988) Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea-pig. J. Physiol., 403: 621-640. Kaila, K. (1994) Ionic basis of GABA, receptor channel function in the nervous system. Prog. Neurobiol., 42: 489-537. Kaila, K. and Voipio, 3. (1987) Postsynaptic fall in intracellular pH induced by GABA-activated bicarbonate conductance. Nature, 330: 163-165. Katzka, D.A. and Morad, M. (1989) Properties of calcium channels in guinea-pig gastric myocytes. J. Physiol., 413: 175-197. Mironov, S.L. and Lux, H.D. (1991) Cytoplasmic alkalinization increases high-threshold calcium current in chick dorsal root ganglion neurones. Ptliigers Arch., 419: 138-143. Moody, W.J. (1984) Effects of intracellular H+ on the electrical properties of excitable cells. Annu. Rev. Neurosci., 7: 257-278. Moody, W.J. and Hagiwara, S. (1982) Block of inward rectification by intracellular H+ in immature oocytes of the starfish Mediasfer aequalis. J. Gen. Physiol., 79: 115-130. Paalasmaa, P., Taira, T., Voipio, J. and Kaila, K. (1994) Extracellular alkaline transients mediated by glutamate receptors in the rat hippocampal slice are not due to a proton conductance. J. Neurophysiol., 72: 2031-2033. Paradiso, A.M., Tsien, R.Y. and Machen, T.E. (1987a) Digital image processing of intracellular pH in gastric oxyntic and chief cells. Nature, 325: 447-450.
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