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Regulation of Axonal pH
CUXRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 22
Regulation of Axonal pH WALTER F . BORON Department of Phy5iolog.y Yule University School of Medicine New Huuen. Connecticut
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Methodology . . . . . . . . . . . . . . . 111. Effect of Weak Bases and Aci , ................................... A . NH3 and NH:
E. Possible Mechanisms of Transport. ........................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
249 252
268
INTRODUCTION
The intracellular pH (pH,) of squid giant axons is of interest for two reasons. In the first place, the squid axon serves as a valuable model of pH, regulation in other systems. Second, because numerous physiological processes are sensitive to changes in pH,, the acid-base physiology of squid axons is of particular interest to those working on this preparation. The intracellular pH of squid axons was first studied by Caldwell (1954, 1958). and later by Spyropoulos (1960), Bicher and Ohki (1972), and Boron and De Weer (l976a). These workers, all of whom used pH-sensitive microelectrodes to measure pH,, established that the normal pH, of squid axons (i.e., -7.3 at about 20°C) is far too high for H+ and/or HCO, to be in electrochemical equilibrium across the axon’s cell membrane. These 249 Copyright 0 1984 by Acddemlc Presa, Inc All righta of reproduction in any form rererved ISBN 0-12-153322-0
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WALTER F. BORON
data corroborated similar conclusions being drawn at about the same time from experiments on other excitable cells (see Caldwell, 1956; Waddell and Bates, 1968; Roos and Boron, 1981). Specifically, the squid axon’s membrane potential (V,) of --60 mV and external pH (pH,) of -7.8 would predict an equilibrium pHi of -6.8, a half pH unit lower than the actual pHi . Clearly, if pHi is to be kept at the relatively alkaline value of 7.3, an active-transport mechanism must continuously extrude acid from the axon as rapidly as the acid accumulates in the cell as the result of cellular metabolism and the passive fluxes of ions. In this article I will first review some of the factors that modulate pHi and then summarize our understanding of the active-transport mechanism responsible for pHi regulation in squid axons.
II. METHODOLOGY
The experiments described in this article were performed in Woods Hole, Massachusetts, on giant axons of the squid Lofigo pealri. The axons were dissected and cleaned according to standard practices and cannulated at both ends in a dialysis chamber (see Fig. 1). The experiments were of four types. In the first, the only additional manipulation was the insertion of two microelectrodes through opposite end-cannulas (Fig. 1, top, but without dialysis tube). The first microelectrode, an exposed-tip pH-sensitive electrode (Hinke, 1967), is illustrated schematically in Fig. 2. This electrode is capable of exceptionally stable and reproducible recordings, although its large size precludes its use in cells smaller than the squid axon. The second microelectrode was an open-tipped reference electrode (tip size, <5 pm), filled with KCI or potassium glutamate (1 M). In a second group of experiments, a dialysis tube as well as the microelectrodes was inserted into the axon (Fig. 1, top). The internal dialysis technique, developed by Brinley and Mullins (1967), permits the control of intraaxonal concentrations of low-molecular-weight ( M , < 1000) solutes. In a third and fourth group of experiments, we measured isotopic fluxes of Nat and CIF. These axons were internally dialyzed, but not impaled with microelectrodes. Effluxes (Fig. 1, middle) were measured by presenting an isotope to the axoplasm via the dialysis fluid (DF) and assaying the radioactivity of the artificial seawater (ASW), which continuously superfused the axon. Influxes were measured by placing an isotope in the ASW and measuring the radioactivity of the collected DF (Fig. I , bottom). The standard ASW was composed of (mM): 425 Na+, 12 K ’ , 10 Ca2+,50 Mg, 542 C1-, and 30 mM buffer 4-12-hydroxyethyl]-I-piperazinepropanesulfonic acid (EPPS) titrated to pH 8.0 (-970
YDROLYZED
TUBING
DIALYSIS TUBING
--L-y---l
END W E L L
DIALYZED REGION
GUARD
&---
-
GUARD
END WELL
S U C T l O N y
------
FIG. I . Internal dialysis apparatus. T o p : pH, experiments. The squid giant axon (400600 bm in diameter) is tied onto a glass cannula at both ends. A length of dialysis tubing (- 140 p n in diameter) is introduced through one cannula, traverses the length of the axon, and exits through the opposite end cannula. In its hydrolyzed region (dashed lines), the tubing is permeable to low-molecular-weight solutes (i.e., M,< 1000). A pH-sensitive microelectrode (see Fig. 2) is inserted through one cannula, and an open-tipped reference (V,) electrode, through the other. Their tips are fewer than one axon diameter apart. The axon's central region, which is continuously circumfused with artificial seawater (ASW), is isolated from the cut ends by Vaseline petroleum jelly seals. In some pH, experiments, no dialysis tubing was present. Middle: Isotopic efflux experiments. **Na or "CI is presented to the axon's interior via the dialysis fluid (DF) and is collected in the ASW. Isotope exiting into the guard region is discarded; it represents a flux from a part of the axon whose composition is not well controlled by dialysis. Bottom: Isotopic influx experiments. The isotope is presented via the ASW and collected in the DF. The guard regions are completely obliterated by Vaseline petroleum jelly seals. This prevents the entry of isotope through membrane surrounding axoplasm not optimally controlled by dialysis. To maximize the collection of isotope that may diffuse laterally toward the end wells, the hydrolyzed region of the dialysis tube is somewhat wider than the central region over which the isotope is presented. (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
252
WALTER F. BORON
PH -sensitive
\
Frc. 2. Protruding tip pH microelectrode. Designed by Hinke (1967). this electrode consists of an insulating lead glass shank (Corning 0120), through which protrudes a cone of pH-sensitive glass (Corning 0150). The two pieces of glass are welded together on a microforge. The outer diameter of the insulating glass is s 125 pm for the terminal 3-4 cni. The pH glass has a diameter of 40-60 pm at the glass seal, and an exposed length of 100-300 pm.
mosmlkg). The standard DF contained (mM): 350 K + , 50 Na+, 7 Mg'+, 150 CIF, 264 glutamate, 210 taurine, 10 N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES), I .0 ethylene glycol bis(p-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), 0.5 phenol red, and 4.0 ATP (950960 mOsm/kg). For further details, consult Boron and De Weer (197ha) and Boron and Russell (1983). 111.
EFFECT OF WEAK BASES AND ACIDS ON pHi
A. NH3 and NH:
When an axon is exposed to a solution containing NH, and NH: (NH; = NH3 + H + ; p K ; 9 . 9 , pHi at first rises rapidly (see Fig. 3A, segment ah) as the highly permeant NH3 enters the cell and then com-
253
REGULATION OF AXONAL pH
6 9L
IS mm
C
FIG. 3. Effect of NHJNH: on pH,. (A) Short-term exposure. The axon is exposed to artificial seawater (ASW) (pH 7.70 throughout) in which 10 mM NaCl has been replaced with NH4CI. The influx of NH3produces an alkalinization as the weak base combines with H' to form NH; (segment ab). When the external NH3/NH; is removed, pH, falls to a value slightly below its initial one (compare c and a). (B) Long-term exposure. When the treatment with NH3/NH; is more prolonged, the initial alkalinization ( c d ) is clearly followed by a slower acidification during a period termed the plateau phase ( d e ) .The subsequent washout of NHJNH; leads to a pH, undershoot of exaggerated size (comparef'and c ) . (C) Proposed mechanism for segment di,plateau-phase acidification. The passive entry of NH: is followed by the intracellular dissociation of a small amount of this weak acid to H + and NH3. The resultant increase of [NH,], leads to a net efflux of NH1. Thus, the NH3/NH: couple shuttles H + into the axon during the exposure to NH,/NH;. (From Boron and De Weer, 1976a; reproduced by permission of the Rockefeller University Press.)
bines with H' to form its conjugate weak acid, NH;. This entry and protonation of NH3 continues until [NH,], reaches [NH,],, at which point the increase in pH, is expected to halt. When the external NH3 and NH; are removed, these processes reverse, and pHi falls ( h c ) ;note, however, that pHi undershoots its initial value by a small amount (compare a and c ) , The magnitude of the NH,-induced intracellular alkalinization is determined by [NH,],, the initial pH, (the law of mass action predicts that the higher the starting pH,, the smaller the subsequent pHi increase), and the
J69
254
WALTER F. BORON
intracellular buffering power (p). Indeed, the change in pHi produced by application or withdrawal of extracellular NH3 is used to calculate /3 (see Boron, 1977); in squid axons (pHi = 7.3-7.8) p = 9 mM (Boron and De Weer, 1976a). Experiments in which the axon is exposed to NH3 and NH: for more prolonged periods indicate that the cell membrane is permeable to NH: as well as to NH,. In the example of Fig. 3B, exposing the axon to NH3 and NH: causes an initial rise in pHi ( c d ) ,followed by a slower decline during a period termed the "plateau phase" (de).Throughout this plateau phase, the NH,f electrochemical gradient favors the influx of this ion.' As NH: passively enters the axon, most2 of it remains NHd and has no immediate effect on pHi. A small fraction, however, dissociates into H+ and NH, and produces a plateau-phase acidification. Because [NH3], rises slightly above [NH,],, NH3 passively leaves the cell. Thus, the oppositely directed fluxes of NH3 and NH: acidify the cell by shuttling protons inward (Fig. 3C). When the NHJNH: is removed from the external solution (point e ) , virtually all intracellular NH: exits the cell as the highly permeant NH3, after having first given up its H+ and lowered pHi (segment ef). This dissociating intracellular NHd includes not only the NH4f derived from the NH3that entered during segment cd, but also the NH: that directly entered the cell during segment de. Note that the magnitude of the pHi undershoot (compare points c andf) is greater than that of the plateau-phase acidification (compare d and e ) , even though both are caused by the influx of NH:. This apparent discrepancy arises because only a small fraction of the entering NH: dissociates during the plateau phase; the rest dissociates only after removal of external NH3/NH:. We have found that the magnitude of the pHi undershoot upon removal of the external NH,/NH,f is directly related to the amount of NH; that had previously entered. Thus, the small pHi undershoot in Fig. 3A reflects the relatively short exposure to NHJNH:. The importance of the NH: electrochemical gradient is evidenced by an experiment in which this gradient is inverted by depolarizing the axon with high [K+],. Not only does pHi rise during the plateau phase (owing to NH: efflux), but removal of external NH:/NH3 produces a pHi shortfall rather than an undershoot (W. F. Boron and P. De Weer, unpublished observation). I At point d in Fig. 3B, [ N H J ] is ~ approximately equal to [NH,],. If the dissociation constant ( K : ) of the reaction NH; = NH3 + H+is the same inside and outside the cell, then it follows that [H'li INH:li = tG lNH31i = K: [NH31, = [H+I,, [NH:],. In other words, the At point d, EHis -0 m V , equilibrium potential for NH: is the same as that for H + (EH). whereas the membrane potential is ---58 mV. Thus, the electrochemical gradients strongly favor the influx of both H +and NH; . * The fraction of the entering NH: which remains NH: is determined by the relationship between the pK: (-9.5) of the reaction NH: = NH3 + H', and the current pH,. At a pHi of 7.7, the ratio of entering NH; which remains NH: to that which dissociates is about 50 : 1.
REGULATION OF AXONAL pH
255
NH3 is by no means the only weak base that produces sizable increases of intraaxonal pH. Methylamine, triethanolamine, and the K+-channel blocker 4-aminopyridine all cause comparable intracellular alkalinizations (W. F. Boron and P. De Weer, unpublished observation). B. COOand HCO;
When an axon is exposed to a C02-containing solution, pHi falls rapidly (see Fig. 4A, segment ab) as C02 enters the cell and combines with H 2 0 to form H2CO3, which then dissociates into H+ and HCO;. CO2
+ H20 = H2CO3 = H' + HCO;
The entry of C02 and the resultant intraaxonal acidification continue until [C0,li = [CO,],. The extent of the intracellular acidification depends on [CO,],, p, and the initial pHi (because C02 + H 2 0 = H+ + HCO,, the higher [H+],the less the tendency for the reaction to proceed to the right). When the C02/HCO; is removed from the external solution, these processes reverse and pHi returns toward its initial level ( c d ) .However, if, during the C 0 2 exposure, pH, is sufficiently high (e.g., 7 . 9 , pHi returns to a value slightly higher than the initial one (compare points a and d ) . This overshoot is caused by the axon's pHi-regulating system (see below). In the experiment of Fig. 4A, the fall of pHi brought about by the application of C02 appears to be sustained (segment bc) only because the C02 exposure is relatively brief. If the exposure is more prolonged (see Fig. 4B), the initial intracellular acidification (ab) is followed by a slow rise in pHi (bc). This alkalinization cannot be accounted for by passive transport processes, because the predicted passive fluxes of H+ (an influx) or HCO, (an efflux) would each tend to further acidify the axon. The slow segment -bc pH, recovery must therefore be caused by an activetransport mechanism that extrudes acid from the cell (Fig. 4C). When the external C02/HC0; is removed, pHi overshoots its initial value (compare a and d ) by an exaggerated amount. The magnitude of this overshoot is a direct reflection of the amount of acid extruded from the axon during the preceding CO, exposure. Thus the overshoot after the 25-min C02 exposure of Fig. 4A is only about 20% as large as the overshoot following the 125-min C 0 2 exposure of Fig. 4B. Although snail neurons (Thomas, 1976b) and barnacle muscle fibers (Boron et al., 1979) can return pHi to normal (provided pH, and [HCO;], are sufficiently high) after even very large intracellular acid loads (i.e., sufficient to lower pHi by 0.5-1.0), squid axons cannot return pHi to normal during an in vitro experiment unless the acid load is quite small
WALTER F. BORON
5 % co2
B 7.4 75[
'
7-
4
I
>
-
/
50
FIG.4. Effect of Cot on pH,. (A) Brief exposure. An intact axon is exposed to ASW containing 5% c o 2 / 5 0mM HCO;; extracellular pH (pH,) is 7.5 throughout. The influx of C 0 2causes pH, to fall (segment ah) and apparently to level off ( h c ) . The subsequent return to a HCO1-free ASW (buffered with 5 mM Tris-HEPES) produces a slight overshoot of the initial pH, (compare d and a ) . (B) Prolonged exposure. In a more lengthy experiment, it is clear that the initial C02-induced fall in pHi (ah)is followed by a slower recovery ( h c ) .When the COz is now washed out, pH, overshoots its initial value by an exaggerated amount (compare d and ( I ) . (C) Proposed mechanism of segment -6c alkalinization. During the plateau phase of the above C 0 2 exposure, the electrochemical gradients for H' and HCO; favor a net influx and efflux, respectively, of these two ions. Thus, the observed increase in pH, must be due to an active transport process. Although depicted here as a proton extruder, this process could involve the uptake of alkali and/or the efflux of acid. The transport process has been generically described as "acid extrusion." (From Boron and De Weer, 1976a; reproduced by permission of the Rockefeller University Press.)
REGULATION OF AXONAL pH
257
(i.e., lowering pHi by -0. I ) . At a pH, of 7.5, a [HCO.;], of 50 mM, and a pHi of 7.0, the axon’s acid-extrusion rate is exceeding low, being able to raise pHi by only -0.1 pH unit per hour (Boron and De Weer, 1976a). After a few hours, the pH, recovery halts even though pHi is far below its normal value. We suspect that long-term metabolic changes either decrease the acid extrusion rate and/or increase the acid-loading rate, thereby preventing a complete pHi recovery. IV.
EFFECT OF METABOLIC INHIBITORS Off pH1
Cyanide (CN-), 2,4-dinitrophenol (DNP), and azide (Ny) all produce a slow and prolonged intracellular acidification that begins after a short delay. In addition, the latter two substances cause a more rapid but brief phase of acidification that begins very soon after they are applied. As illustrated in Fig. 5A, cyanide has little effect on pHi for the first 5 min, after which it causes pH, to decline by -0.3 over the next -40 min (Boron and De Weer, 1976a). The lack of an immediate acidification implies that the influx of the neutral weak acid (HCN = H+ + CN-) is not followed by a substantial dissociation into H + and CN-. Indeed, the dissociation is expected to be very small because HCN has such a high pK; (-10). The slow fall in pHi is likely the result of a buildup of acidic metabolites,
FIG.5 . (A) Effect of cyanide on pH, and V , . Exposing the axon to ASW containing 2 mM NaCN (pH, = 7.70) causes, after a 5-rnin delay, a slow fall of pH, of -0.3. Simultaneously, there is a transient depolarization, followed by a sustained hyperpolarization. The effect of CN is only partially reversible in the first few minutes. (B) Effect of azide on pH, and V,. Application of 3 mM NaN, (pH, = 7.70) causes a rapid internal acidification followed, after a brief delay, by a slower pH, decline. The membrane voltage undergoes a transient depolarization. followed by a sustained hyperpolarization. The changes are only partially reversible in the first several minutes. (From Boron and De Weer, 1976a; reproduced by permission of the Rockefeller University Press.)
258
WALTER F. BORON
following inhibition of oxidative phosphorylation. It is unlikely to be due to the shuttling of protons across the cell membrane by HCN/CN-, inasmuch as [CN-Ii is exceedingly low (-2 p M ) . Removal of cyanide produces only a small pHi recovery over the first several minutes. The application of azide (3 mM, pH, = 7.70) causes an abrupt fall in pHi of -0.1 (see Fig. SB), followed by a period of stability. If the azide is removed during this period, pHi rapidly returns to its initial value (not shown). Thus, the rapid azide-induced acidification is probably the result of the influx and dissociation of the neutral weak acid (HN3 = Ht + N;; pKA = 4.7). During lengthier exposures to azide, pHi eventually begins a slow decline, similar to the slow phase of acidification produced by cyanide. This slow phase of acidification is likely caused by the build-up of acidic metabolites, the result of azide's uncoupling of oxidative phosphorylation. The inward shuttling of protons across the cell membrane by the HN3/N: couple is probably not a major contributor to this acidification, inasmuch as the slow pHi fall begins only after a lengthy delay, and because reversing the N; electrochemical gradient has only a small effect on pHi (Boron and De Weer, 1976a). Removal of the azide causes a rapid pHi recovery, but only to the same extent as the initial, rapid phase of azide-induced acidification. 2,4-Dinitrophenol (1 mM, pH, = 7.70) has an effect similar to that of azide, except that the slow phase of acidification follows the rapid, initial one without an appreciable delay. V.
IONIC MECHANISM OF pH1 REGULATION
The existence of an axonal pHi-regulating mechanism is indicated by the relatively high value for steady-state pHi (i.e., -7.3 vs the equilibrium pHi of -6.8) and by the plateau-phase recovery of pHi from a C02-induced acid load (see Fig. 4B). These results suggest that an axonal pHi regulator responds to intracellular acid loads by extruding acid from the cell. Detailed mechanistic information can be gained by studying the recovery of pHi from acute, intracellular acid loads. A. Dependence on External HCO;
In the experiment of Fig. 4B, the recovery of pHi from the COJnduced acid load necessarily took place in the presence of external HCO,. The experiment of Fig. 6 was designed to test whether HCO; is required for the recovery of pHi from an acute acid load (Boron and De Weer, 1976b). The acid load is applied by pretreating the axon with an ASW containing 50 mM NH: (pH, = 7.7). As described above, the application of the NH3/
259
REGULATION OF AXONAL pH
F FIG.6 . Effect of external HCO; on recovery of pH, from an acid load. The axon is acid loaded by pretreating it with artificial seawater (ASW) containing 50 mM NH: (pH, = 7.7), and then washing out the NHJNH:. As shown in Fig. 3B, the washout causes pH, to undershoot its initial value; i.e., the cell is acid loaded. In the nominal absence of HCO; (segment AB), pH, fails to recover. During the application of ASW containing 10 mM HCO; at the same pH,, however, pH, recovers more rapidly (segment C D ) . After washout of the CO,/HCO;, pH, no longer recovers (point E ) . (From Boron and De Weer, 1976b; reproduced by permission of Macmillan Journals, Ltd.)
NH: causes an abrupt increase in pHi (due to NH3 influx) followed by a slower fall (due to NH; influx). When the external NH3/NH: is washed out, pHi falls to a value substantially lower than its initial one (i.e., the cell is acid loaded). In the nominal absence of HCO, (segment AB), pHi recovers only very slowly, even though pH, is 8. The application of ASW containing 0.4% C 0 2 and 10 mM HCO; at constant pH, causes a transient acidification (BC) due to the influx of C 0 2 .This is followed, however, by a marked acceleration of the pHi recovery (compare CD and AB). This suggests that external HCO; is required for acid extrusion in the squid axon, The rate of the segment -CD recovery can be used to calculate the rate of acid extrusion (i.e., the equivalent net HCO; influx, JFbo3). This is simply the product of the recovery rate (dpHJdt), intracellular buffering power (p),and axonal volume-to-surface ratio ( p ) . A more extensive series of experiments (Boron and Russell, 1983), similar in design to the experiment of Fig. 6 (i.e., in nondialyzed axons),
260
WALTER
KINETICS OF
External HCO, External Na' Internal CIP
F. BORON
TABLE I AXON'SpH,-RECUt.ATING
THE SQUID
SYSTEMS''
30 21 38
2.3 +- 0.2 77 -t 13 84 f 15
10.6 2 0.4 10.3 t 0.6 19.6 1.2
*
Kinetic parameters obtained from nonlinear, leastsquares curve fit. n is number of data points (measurements of net, equivalent HCO, flux); V:,,, is apparent maximal velocity; KL, is substrate concentration at half VLdx. Standard conditions: [Na'], = 425-437 mM, [HCO;], = 12 mM, pH, = 8.0, [el-], = 100 m M , pH, = 6.7.
has shown that the dependence of acid-extrusion rate on [HCO,], follows Michaelis-Menten kinetics (see Table I). The apparent K , for external HCOT is -2.3 mM (pH, = 8.0, pHi = 6.7, T = 22°C) and the apparent VmaXis 10.6 pmol ~ r n sec-' - ~ (at a physiological [CI -Ii of 70-100 mM). The magnitude of the acid-extrusion rate has been carefully documented (Boron and Russell, 1983) in a series of experiments on internally dialyzed axons. Isotopically measured fluxes of Na+ and C1- (see below) were determined under identical conditions (pHi = 6.7, [ClFIi= 150 mM, pH, = 8.00, [HCO;], = 10 mM, (Na'l, = 425 mM, T = 16°C) for the purpose of determining the stoichiometry of acid extrusion. The approach in the acid-extrusion rate experiments was to dialyze the axon with a lowpH fluid until pHi fell to -6.7, at which time dialysis was halted, thereby returning control of pHi to the axon. After dialysis was halted, pHi failed to recover (i.e., increase) until HCOC was added to the external solution. From thc rate of HCOi-stimulated pHi recovcry, axon diameter, and buffering power,' the acid-extrusion rate (J$io,) was computed. In 15 axons, the mean JnHeEOjwas 7.5 pmol cm12 sec-' (see Table 11). This acid extrusion was completely blocked by the anion-flux inhibitor 4-isothiocyano-4'acetamido -2,2'-disulfonate (SITS), as had previously been demonstrated The non-C02 or intrinsic buffering power (PI)was determined by halting dialysis, blocking the pHi,-regulating systems with SITS, and then exposing the axon to ASW containing 0.2 mM NH; at pH 7.75 (see Boron, 1977; Boron and Russell, 1983). PI, the quotient of the calculated change in [NH;], and the measured pHi change, came to 11.2 mM. The total buffering power (&) is the sum of PI and the COz buffering power (PcOz).The latter is calculated from the relation pco, = 2.3 [HCO, Ii,and comes to I .7 mM under the conditions of these experiments. Thus, PT = 11.2 + 1.7 = 12.9 mM.
REGULATION
OF AXONAL
261
pH
TABLE I1
AXON'S ~H,-RECUI.ATING SYSTEM"
STOICHIOMETRY OF THE SQUID
Ion
Na' CIHCO;
Influx
Efflux
3.4 t 0.4 (13) 0.1 t 0.2 (6) -
0.0 t 0.1 (6) 3.9 t 0.2 (15)
-
Net flux (pmol cm sec-I) +3.4
-3.8
1.5
%
0.6
(15)
Mean kSE, with number of experiments given in parentheses. The HCOj flux was calculated from the rate of pH, recovery and I S the net, equivalent HC03 flux referred to in the text. Standard conditions: temperature = 16°C. [Nail, = 425 mM. [HCO,] = 12 mM, pH, = 8.0, [CI 1, = 150 mM, pH, = 6.5.
for this preparation (Russell and Boron, 1976), as well as the snail neuron (Thomas, 1976a) and barnacle muscle (Boron, 1977). 6. Dependence on External Nai
In 1977, Thomas showed that acid extrusion in snail neurons requires external Na+, an observation confirmed on giant barnacle muscle fibers (Boron et ul., 1979). The experiment of Fig. 7 demonstrates how the recovery of pH, from an NHJNH4f-induced acid load depends on "a+], in the squid axon (Boron and Russell, 1983).After removal of external NHl/ NH;, pH, is allowed to fall and level off (segment ab) in a nominally HC0;-free ASW. Application of 10 mM HCO; ASW containing 425 mM Na+ (bc)causes a rapid recovery of pH, (calculated JFbo3 = 9.7 pmol cm-2 sec-I). The pH, recovery is slowed (cd) by reducing "a+], to 15 mM (JE& = 1.1 pmol cm-2 sec-I), and then accelerated (de) by raising "a+], to 100 mM (J$& = 6.4 pmol cm-2 sec-I). The results of a series of such experiments showed that the dependence of acid-extrusion rate on "a+], follows Michaelis-Menten kinetics, with an apparent K , of 77 mM (pH, = 8.00, pH, = 6.7, temperature = 22°C). Not only does acid extrusion require external Na+, but it is accompanied by a net influx of Na+. Unidirectional Na+ effluxes and influxes (measured with 22Na)were determined in experiments in which axons were dialyzed to a pH, of 6.7 and a [CI-1, of 1.50 m M .The pH,-regulating system-linked Na+ flux was defined as the increase in the Na+ flux pro-
262
WALTER F. BORON klOO N$ PHO 7 7 0
,
12 HCOi
[Notlo:
425
pHo 8.00 IS I00
Fie. 7 . Dependence of acid-extrusion rate on [Na+l0.The axon was acid loaded by pretreating it with artificial seawater (ASW) containing 100 mM NH: (pH, = 7.70). After washout of the NH1/NH:, pH, failed to recover from the acid load (segment ub) until HCO; was added to the ASW. The subsequent pH, recovery was rapid when "a'], was 425 mM ( h c ) , but it slowed considerably when "a+], was reduced to 15 mM ( c d ) . Raising "a+], to 100 mM (de) brought the pH, recovery rate to a value intermediate between the first two values. (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
duced by the application of 10 mM HCO, (pH, = 8.0) ASW. We found that, under standard conditions, the unidirectional Na+ efflux mediated by the axon's pHi-regulating system is approximately zero, and that the influx is about 3.4 pmol cm-2 sec-' (Boron and Russell, 1983). Thus, the pHi-regulating system mediates a net Na' influx (see Table 11). This net Na+ influx shares all the properties of the HCO: flux: (1) it is blocked by SITS; (2) it requires internal C1-; and (3) it requires internal ATP. The
263
REGULATION OF AXONAL pH pH 6.6 k 2 0 0 CI-
Inside:
'
pH, 8.00
'
Outside:
12 HCOj
I
5:"l
7.4
7.2
350 CI-
pHi 7.0
6.8
6.6
30 rnin
FIG.8. Dependence of acid-extrusion rate on [CI-1, . The axon was loaded by predialyzing (segment ab) it with a fluid at pH 6.6, containing 200 mM C1-. When dialysis was halted, returning control of pH, to the axon ( b c ) ,there was no pH, recovery until 12 m M HCO, was added to the artificial seawater (cd). The inset shows the results of three similar experiments (during segments bc and c d ) in which the axons were dialyzed to [CI-1, levels of 0, 100, and 350 mM CI-. (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
magnitude of the net Na+ influx is about 0.45 as large as the J;;CO3obtained under identical conditions. This is consistent with a HCO, : Na+ stoichiometry of 2 : 1. C. Dependence on Internal CI-
When internal CI- is removed by dialyzing the axon with a C1-free solution, acid extrusion is blocked (Russell and Boron, 1976). This dependence on internal CI- is demonstrated by the experiment of Fig. 8 . The axon is dialyzed with a low-pH solution containing 200 mM CIF (segment ab). After dialysis is halted, returning control of pHi to the axons, there is no pHi recovery (bc) until HCOj is added to the external solution. The HCOy-stimulated rise in pHi (at the beginning of the segment cd pHi recovery) corresponds to an acid-extrusion rate of 15.0 pmol cm-2 sec-I. The inset shows three additional experiments (each on a different axon) at [Cl-Ii values of 0, 100, and 350 mM. It is clear that the pHi-recovery rate gradually increases as [ClFIi is raised. The results of 38 similar experi-
264
WALTER
F. BORON
ments show that the dependence of acid-extrusion rate on [C1-Ii follows Michaelis-Menten kinetics, with an apparent K , for internal C1- of 84 mM (see Table I). This value is in the range of reported normal values of [CIVliin squid axons (Brinley and Mullins, 1965) and indicates that the axon's pHi-regulating system is normally only about half saturated with respect to intracellular C1- . This is in marked contrast to the other two substrates (i.e., external Na+ and HCO,), for which the transporter is nearly saturated under physiological conditions. The dependence of acid extrusion on internal C1- has also been demonstrated for snail neurons by Thomas (1977). Acid extrusion not only has an absolute dependence on internal C1-, it is accompanied by a net CIV efflux. When the pHi of dialyzed axon is lowered to 6.7 under the aforementioned standard conditions (pHi = 6.7, [Cl-li = 150 mM, pH, = 8.00, [HCO,], = 10 mM, "a+], = 425 m M , temperature = 16°C) the addition of 10 mM HCO, to the ASW has minimal effect (stimulation of -0. I pmol ern--* sec - I ) on the unidirectional CIinflirx (see Table II), measured with "CI. The CIV c f f h x , however, increases by about 3.9 pmol cm-2 sec-'. The net CI- efflux, 3.8 pmol cm-* sec-I, is 51% as large as the net equivalent HCOY flux, but is approximately the same as the net N a + influx. The external HCOy-stimulated C1efflux has all the properties expected of a flux tightly linked to the pHiregulating system (Boron and Russell, 1983): it ( I ) is blocked by SITS, (2) requires external Na+, (3) requires internal ATP, and (4) is stimulated by IOW pHi. D. Dependence on Internal ATP
If squid axons are treated with either cyanide (2 mM) or DNP (1 mM), pHi fails to recover from a C02-induced acid load. This inhibition is reversible in both cases (Boron and De Weer, 1976b). Although Caldwell (1960) had previously shown that treatment with cyanide or DNP reversibly lowers [ATPI, substantially, the above experiments left open the possibility that cyanide and DNP inhibit the pHi regulator directly, not through their effect on ATP levels. The experiment of Fig. 9 was designed to determine whether, in the continuous presence of cyanide, the addition of ATP would restore the axon's ability to recover from an acid load. In the first part of the experiment, the axon is dialyzed with an ATP-free solution for 1 hr; the dialysate's pH is lowered to 6.6 during the final 30 min to acid load the axon. When dialysis is halted (point a ) , returning control of pH, to the axon, there is no pHi recovery (segment u b ) , even
265
REGULATION OF AXONAL pH INTERNAL FLUID
-
ATP fro. pH Z3
'
pH 6.6
EXTERNAl FLUID
~
8mMATP pH 7.3' pH 6.6
, HC03
FIG.9. Dependence of acid extrusion on ATP. The axon was exposed throughout to artificial seawater containing 2 mM CN. Predialysis with an ATP-free fluid at pH 7.3 was followed with a similar fluid at pH 6.6. After pHi fell to -6.8, dialysis was halted, returning control of pH, to the axon. There was no pH, recovery, however, either in the absence (segments ah and c d ) or in the presence of HCO;. After ATP was reintroduced to the axon (de), in the continued presence of ATP, the application of HCO, elicited a pH, recovery (fg) which was blocked by SITS ( g h ) .(From Russell and Boron, 1976; reproduced by permission of Macmillan Journals. Ltd.)
during stimulation with HCO.7 (bc). After the axon is dialyzed with an ATP-containing solution ( d e ) ,however, application of HCO, does elicit a recovery of pHi from the acid load ( f g ) , and this recovery is blocked by SITS ( g h ) . Thus, ATP or a related substance is required for acid extrusion. As noted above, ATP is also required for the net Na+ influx and C1efflux that are coupled to acid extrusion. Although ATP is necessary for transport, it is not clear whether hydrolysis of ATP is stoichiometrically linked to transport, or whether ATP merely fills a catalytic role. E. Possible Mechanisms of Transport
The above data indicate that the squid axon possesses a SITS-sensitive ion-transport mechanism that responds to intracellular acid loads by extruding acid from the cell. The transport not only requires external HCO? and Na+ and internal C1-, but is accompanied by a net Na+ influx and a net C1- efflux. The data are consistent with a stoichiometry of one Na+ entering the axon for each C1- exiting, and for every two acid equivalents
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WALTER F. BORON
neutralized inside the axon (see Table 11). The implication of the stoichiometry data that transport is electrically neutral is corroborated by the observation that changes in membrane potential are without significant effect on the acid extrusion rate. These data are accounted for by each of the models of Fig. 10. The first model (Thomas, 1977) has Nat and HCOj entering in exchange for C1- and H+. In the second, the exit of H+ is replaced by the entry of a second HCO,. In the third model, the entry of a single C0:- replaces that of two HCOT. Finally, according to the last model (Becker and Duhrn, 1978; Boron, 1980), Na+ and COi- enter only after forming the ion pair NaCOT (Garrels et al., 1961). These models are equivalent thermodynamically. The free-energy change associated with the overall transport process (AGnec)is the sum of the individual freeenergy changes of the transported ions. In the following example, these free-energy changes are computed for the relatively low pHi of 6.7, assuming normal values for [Cl-]; and “a+];. The calculations are based on the first model of Fig. 10. OUT
IN
FIG.10. Models of acid extrusion. The models are equivalent thermodynamically. They all predict electroneutral transport with a stoichiometry equivalent to one NaCtaken up for each C1- lost, and for each two protons neutralized inside the cell tie., by the uptake of HCO; or a related species and/or the efflux of H+). (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
267
REGULATION OF AXONAL pH
AGNa = RT In(INa+],/INa+l,) RT In([HCOi],/[HCOj],) ACcl = RT In([CI-l,/[CI 1,) AGH = RT In([H+],/[H+],)
AGHCo, =
RT ln(50/425) RT ln(0.5/10) = RT ln(550/100) = RT ln(10'8/10-6 7, =
=
=
= = =
-2.14RT -3.00RT +1.70RT -3.00 RT
Thus, the overall free-energy change of the acid-extrusion reaction (pHi = 6.7) is -6.44 RT, the negative value indicating that the reaction ought to proceed spontaneously as written (i.e., extruding acid from the cell). At higher pHi values the calculated AG,,, is less negative (i.e., [H+Iifalls and [HCOjli rises). However, even at the normal pHi 7.3, where the observed is greatly reduced and the accompanying Na+ and C1- fluxes are undetectably low, the AG,,, is still -3.66 RT, and thus still favors the net forward reaction by a wide margin. A similar relationship among AGnet,acid extrusion, and pHi exists in barnacle muscle. In this cell, pHi is regulated by a Na/HCO,-Cl/H exchanger similar to that of the squid axon. The acid-extrusion rate in barnacle muscle falls to near zero at the pHi of 7.3-7.4, even though there is sufficient energy in the ion gradients to drive pHi to -8. The barnacle's pHi-regulating system also mediates bidirectional C1- fluxes and bidirectional Na+ fluxes. Interestingly, all these fluxes are blocked at pHi values greater than -7.3 (Boron et al., 1979; Russell et al., 1983). Thus, all known modes of ion transport by the pHi regulator are blocked at pHi values exceeding 7.4. It is unlikely that the inhibition of all modes of transport at pHi > 7.4 is due solely to unavailability of substrate (i.e., intracellular H+). Indeed, if the apparent transporter-mediated C1- influx and Na+ efflux were due to the microscopic reversibility of the pHi regulator, then one might expect low [H+Iito stimulate rather than inhibit these fluxes. It would thus appear that there is an allosteric site for H+ on the internal surface of the barnacle muscle's pHi regulator. We would predict that this site must be protonated in order for the transporter to operate in any mode. Inasmuch as the squid axon's pHi regulator does not appear to mediate an appreciable Cl- influx or Na+ efflux during acid extrusion (see Sections V,B and V,C), it will be difficult to establish the presence or the absence of a similar allosteric H+ site in this preparation. It is interesting to note that the Na-H exchanger, which regulates pHi in several vertebrate cell types, has a pHi dependence that is similar to that of the Na/ HCO,-Cl/H system (see Aickin and Thomas, 1977; Boron and Boulpaep, 1982a), which suggests that it may have an internal allosteric site for H+. This suspicion has been confirmed by the vesicle studies of Aronson et al. (1982).
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Concerning the ATP dependence of the squid axon’s pHi regulator, the preceding thermodynamic calculations point out that there is more than sufficient energy in the gradients of the transported ions to account for the activity of the pH, regulator. If the ion gradients were the only source of energy for the transporter, then one might expect transport to bc reversed by appropriately inverting the gradient of one or more of the transported ions. When such gradient inversions are imposed on the barnacle muscle’s Na/HCO,-CI/H exchanger, for example, transport clearly reverses (Russell et ul., 1983). Reversal of the pH, regulator, however, has yet to be demonstrated in squid axons, despite considerable effort (Boron, unpublished). The squid axon pHi regulator’s dependence on ATP and its resistance to reversal could be explained by a tight coupling of transport to ATP hydrolysis. However, we cannot yet rule out the possibility that ATP is required merely for catalytic purposes, and that reversal of acid extrusion is difficult to observe only for kinetic reasons. Although the four models of Fig. 10 are equivalent thermodynamically, they do not necessarily make the same kinetic predictions. In particular, the ion-pair model predicts that the acid-extrusion rate should not depend on “a’],, or [HCO;], per se, but on [NaCOj] at a given pH. Indeed, the Na+ and HC03- data of Table I indicate that whether [Na ‘I, is varied at a constant [HCOj], of 12 mM or [HCOI], is varied at a constant “a+] of 425 mM, the acid-extrusion rates fall on the same velocity vs [NaCO;], curve. The apparent K , for NaCOr is 74 W M (Boron and Russell, 1983). More recently, these studies have been expanded to include the [Na’], dependence of acid extrusion at three [HCOI] levels (12, 6 , and 3 mM), and the [HCOj], dependence at three “a+] levels (425, 212, and 106 rnM) (Boron, unpublished). In all cases, the data fall on the same velocity vs [NaCOy1, curve, in agreement with the ion-pair model. REFERENCES Aickin, C . C., and Thomas, R. C. (1977). An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers. J . Physiol. (London) 273, 295-3 16. Aronson, P . S . , Nee, J . , and Suhm, M. A. (1982). Modifier role of internal H’ in activating the Na-H exchanger in renal microvillus vesicles. Nature (London) 299, 161-163. Recker, B . F., and Duhm, J. (1978). Evidence for anionic cation transport of lithium, sodium and potassium across the human erythrocyte membrane induced by divalent anions. J . Physio/. (London) 282, 149- 168. Bicher, H . , and Ohki, S. (1972). Intracellular pH electrode experiments on the giant squid axon. Biochirn. Biophys. Aria 255, 900-904. Boron, W. F. (1977). lntracellular pH lransients in giant barnacle muscle fibers. A m . J . Physiol. 233, C61-C73. Boron, W. F. (1980). Intracellular pH regulation. Curr. T ( J ~ Membr. . Trcinsp. 13, 3-22.
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Boron, W. F., and Boulpaep, E. L. (1982a). Intracellular pH regulation in the renal proximal tubule of the salamander: Na-H exchange. J . Gen. Physiol. 81, 29-52. Boron, W. F., and Boulpaep, E. L. (1982b). lntracellular pH regulation in the renal proximal tubule of the salamander: Basolateral HCO, transport. J . Gen. Physiol. 81, 53-94. Boron, W. F., and De Weer, P. (1976a). Intracellular pH transients in squid giant axons caused by C 0 2 , NH,, and metabolic inhibitors. J . Gen. Physiol. 67, 91-112. Boron, W. F.. and Ile Weer. P. (1976b3. Active proton transport stimulated by CO?/HCO:r. blocked by cyanide. Nature (London) 259, 240-241. Boron, W. F., and Russell, J . M. (1983). Stoichiometry and ion dependencies of the intracelMar-pH-regulating mechanism in squid giant axons. J . Gen. Physiol. 81, 373-399. Boron, W. F., McCormick, W. C., and Roos, A. (1979). pH regulation in barnacle muscle fibers: Dependence on intracellular and extracellular pH. A m . J . Physiol. 237, C185C193. Brinley, F. J., and Mullins, L. J. (1965). Variations in the chloride content of isolated squid axons. Physiologisl 8, 121. Brinley, F. J . , and Mullins, L. J. (1967). Sodium extrusion by internally dialyzed squid axons. J . Gen. Physiol. 50, 2303-233 I. Caldwell, P. C. (1954). An investigation of the intracellular pH of crab muscle fibers by means of microglass and micro-tungsten electrodes. J . Physiol. (London) 126, 169-180. Caldwell, P. C. (1956). lntracellular pH. Int. Reu. Cyrol. 5, 229-277. Caldwell, P. C. (1958). Studies on the internal pH of large muscle and nerve fibers. J . Physiol. (London) 142, 22-62. Caldwell, P. C. (1960). The phosphorus metabolism of squid axons and its relationship to the active transport of sodium. J . Physiol. (London) 152, 545-560. Garrels, R. M., Thompson, M. E., and Siever, R. (1961). Control of carbonate solubility of carbonate complexes. A m . J . Sci. 269, 24-45. Hinke, J . A. M. (1967). Cation-selective microelectrodes for intracellular use. In “Glass Electrodes for Hydrogen and Other Cations” (G. Eisenman, ed.), 464-477. Dekker, New York. Roos, A., and Boron, W. F. (1981). lntracellular pH. Physiol. Rev. 61, 296-434. Russell, J. M . , and Boron, W. F. (1976). Role of chloride transport in regulation of intracellular pH. Nature (London) 264, 73-74. Russell, J . M., Boron, W. F., and Brodwick. M. S. (1983). lntracellular pH and Na fluxes in barnacle muscle with evidence for reversal of the ionic mechanism of intracellular pH regulation. J . Gen. Physiol. 82, 47-78. Spyropoulos, C. S. (1960). Cytoplasmic pH of nerve fibers. J . Neurochem. 5 , 185-194. Thomas, R. C. (1976a). Ionic mechanism of the H + pump in a snail neurone. Nature (London) 262, 54-55. Thomas, R. C. (1976b). The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J . Physiol. (London) 255, 715-735. Thomas, R . C. (1977). The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurons. J . Physiol. (London) 273, 317-338. Waddell, W . J . , and Bates, R. G. (1968). Intracellular pH. Physiol. Reu. 49, 285-329.
Regulation of Axonal pH WALTER F . BORON Department of Phy5iolog.y Yule University School of Medicine New Huuen. Connecticut
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Methodology . . . . . . . . . . . . . . . 111. Effect of Weak Bases and Aci , ................................... A . NH3 and NH:
E. Possible Mechanisms of Transport. ........................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
249 252
268
INTRODUCTION
The intracellular pH (pH,) of squid giant axons is of interest for two reasons. In the first place, the squid axon serves as a valuable model of pH, regulation in other systems. Second, because numerous physiological processes are sensitive to changes in pH,, the acid-base physiology of squid axons is of particular interest to those working on this preparation. The intracellular pH of squid axons was first studied by Caldwell (1954, 1958). and later by Spyropoulos (1960), Bicher and Ohki (1972), and Boron and De Weer (l976a). These workers, all of whom used pH-sensitive microelectrodes to measure pH,, established that the normal pH, of squid axons (i.e., -7.3 at about 20°C) is far too high for H+ and/or HCO, to be in electrochemical equilibrium across the axon’s cell membrane. These 249 Copyright 0 1984 by Acddemlc Presa, Inc All righta of reproduction in any form rererved ISBN 0-12-153322-0
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WALTER F. BORON
data corroborated similar conclusions being drawn at about the same time from experiments on other excitable cells (see Caldwell, 1956; Waddell and Bates, 1968; Roos and Boron, 1981). Specifically, the squid axon’s membrane potential (V,) of --60 mV and external pH (pH,) of -7.8 would predict an equilibrium pHi of -6.8, a half pH unit lower than the actual pHi . Clearly, if pHi is to be kept at the relatively alkaline value of 7.3, an active-transport mechanism must continuously extrude acid from the axon as rapidly as the acid accumulates in the cell as the result of cellular metabolism and the passive fluxes of ions. In this article I will first review some of the factors that modulate pHi and then summarize our understanding of the active-transport mechanism responsible for pHi regulation in squid axons.
II. METHODOLOGY
The experiments described in this article were performed in Woods Hole, Massachusetts, on giant axons of the squid Lofigo pealri. The axons were dissected and cleaned according to standard practices and cannulated at both ends in a dialysis chamber (see Fig. 1). The experiments were of four types. In the first, the only additional manipulation was the insertion of two microelectrodes through opposite end-cannulas (Fig. 1, top, but without dialysis tube). The first microelectrode, an exposed-tip pH-sensitive electrode (Hinke, 1967), is illustrated schematically in Fig. 2. This electrode is capable of exceptionally stable and reproducible recordings, although its large size precludes its use in cells smaller than the squid axon. The second microelectrode was an open-tipped reference electrode (tip size, <5 pm), filled with KCI or potassium glutamate (1 M). In a second group of experiments, a dialysis tube as well as the microelectrodes was inserted into the axon (Fig. 1, top). The internal dialysis technique, developed by Brinley and Mullins (1967), permits the control of intraaxonal concentrations of low-molecular-weight ( M , < 1000) solutes. In a third and fourth group of experiments, we measured isotopic fluxes of Nat and CIF. These axons were internally dialyzed, but not impaled with microelectrodes. Effluxes (Fig. 1, middle) were measured by presenting an isotope to the axoplasm via the dialysis fluid (DF) and assaying the radioactivity of the artificial seawater (ASW), which continuously superfused the axon. Influxes were measured by placing an isotope in the ASW and measuring the radioactivity of the collected DF (Fig. I , bottom). The standard ASW was composed of (mM): 425 Na+, 12 K ’ , 10 Ca2+,50 Mg, 542 C1-, and 30 mM buffer 4-12-hydroxyethyl]-I-piperazinepropanesulfonic acid (EPPS) titrated to pH 8.0 (-970
YDROLYZED
TUBING
DIALYSIS TUBING
--L-y---l
END W E L L
DIALYZED REGION
GUARD
&---
-
GUARD
END WELL
S U C T l O N y
------
FIG. I . Internal dialysis apparatus. T o p : pH, experiments. The squid giant axon (400600 bm in diameter) is tied onto a glass cannula at both ends. A length of dialysis tubing (- 140 p n in diameter) is introduced through one cannula, traverses the length of the axon, and exits through the opposite end cannula. In its hydrolyzed region (dashed lines), the tubing is permeable to low-molecular-weight solutes (i.e., M,< 1000). A pH-sensitive microelectrode (see Fig. 2) is inserted through one cannula, and an open-tipped reference (V,) electrode, through the other. Their tips are fewer than one axon diameter apart. The axon's central region, which is continuously circumfused with artificial seawater (ASW), is isolated from the cut ends by Vaseline petroleum jelly seals. In some pH, experiments, no dialysis tubing was present. Middle: Isotopic efflux experiments. **Na or "CI is presented to the axon's interior via the dialysis fluid (DF) and is collected in the ASW. Isotope exiting into the guard region is discarded; it represents a flux from a part of the axon whose composition is not well controlled by dialysis. Bottom: Isotopic influx experiments. The isotope is presented via the ASW and collected in the DF. The guard regions are completely obliterated by Vaseline petroleum jelly seals. This prevents the entry of isotope through membrane surrounding axoplasm not optimally controlled by dialysis. To maximize the collection of isotope that may diffuse laterally toward the end wells, the hydrolyzed region of the dialysis tube is somewhat wider than the central region over which the isotope is presented. (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
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WALTER F. BORON
PH -sensitive
\
Frc. 2. Protruding tip pH microelectrode. Designed by Hinke (1967). this electrode consists of an insulating lead glass shank (Corning 0120), through which protrudes a cone of pH-sensitive glass (Corning 0150). The two pieces of glass are welded together on a microforge. The outer diameter of the insulating glass is s 125 pm for the terminal 3-4 cni. The pH glass has a diameter of 40-60 pm at the glass seal, and an exposed length of 100-300 pm.
mosmlkg). The standard DF contained (mM): 350 K + , 50 Na+, 7 Mg'+, 150 CIF, 264 glutamate, 210 taurine, 10 N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES), I .0 ethylene glycol bis(p-aminoethyl ether)-N,N'-tetraacetic acid (EGTA), 0.5 phenol red, and 4.0 ATP (950960 mOsm/kg). For further details, consult Boron and De Weer (197ha) and Boron and Russell (1983). 111.
EFFECT OF WEAK BASES AND ACIDS ON pHi
A. NH3 and NH:
When an axon is exposed to a solution containing NH, and NH: (NH; = NH3 + H + ; p K ; 9 . 9 , pHi at first rises rapidly (see Fig. 3A, segment ah) as the highly permeant NH3 enters the cell and then com-
253
REGULATION OF AXONAL pH
6 9L
IS mm
C
FIG. 3. Effect of NHJNH: on pH,. (A) Short-term exposure. The axon is exposed to artificial seawater (ASW) (pH 7.70 throughout) in which 10 mM NaCl has been replaced with NH4CI. The influx of NH3produces an alkalinization as the weak base combines with H' to form NH; (segment ab). When the external NH3/NH; is removed, pH, falls to a value slightly below its initial one (compare c and a). (B) Long-term exposure. When the treatment with NH3/NH; is more prolonged, the initial alkalinization ( c d ) is clearly followed by a slower acidification during a period termed the plateau phase ( d e ) .The subsequent washout of NHJNH; leads to a pH, undershoot of exaggerated size (comparef'and c ) . (C) Proposed mechanism for segment di,plateau-phase acidification. The passive entry of NH: is followed by the intracellular dissociation of a small amount of this weak acid to H + and NH3. The resultant increase of [NH,], leads to a net efflux of NH1. Thus, the NH3/NH: couple shuttles H + into the axon during the exposure to NH,/NH;. (From Boron and De Weer, 1976a; reproduced by permission of the Rockefeller University Press.)
bines with H' to form its conjugate weak acid, NH;. This entry and protonation of NH3 continues until [NH,], reaches [NH,],, at which point the increase in pH, is expected to halt. When the external NH3 and NH; are removed, these processes reverse, and pHi falls ( h c ) ;note, however, that pHi undershoots its initial value by a small amount (compare a and c ) , The magnitude of the NH,-induced intracellular alkalinization is determined by [NH,],, the initial pH, (the law of mass action predicts that the higher the starting pH,, the smaller the subsequent pHi increase), and the
J69
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WALTER F. BORON
intracellular buffering power (p). Indeed, the change in pHi produced by application or withdrawal of extracellular NH3 is used to calculate /3 (see Boron, 1977); in squid axons (pHi = 7.3-7.8) p = 9 mM (Boron and De Weer, 1976a). Experiments in which the axon is exposed to NH3 and NH: for more prolonged periods indicate that the cell membrane is permeable to NH: as well as to NH,. In the example of Fig. 3B, exposing the axon to NH3 and NH: causes an initial rise in pHi ( c d ) ,followed by a slower decline during a period termed the "plateau phase" (de).Throughout this plateau phase, the NH,f electrochemical gradient favors the influx of this ion.' As NH: passively enters the axon, most2 of it remains NHd and has no immediate effect on pHi. A small fraction, however, dissociates into H+ and NH, and produces a plateau-phase acidification. Because [NH3], rises slightly above [NH,],, NH3 passively leaves the cell. Thus, the oppositely directed fluxes of NH3 and NH: acidify the cell by shuttling protons inward (Fig. 3C). When the NHJNH: is removed from the external solution (point e ) , virtually all intracellular NH: exits the cell as the highly permeant NH3, after having first given up its H+ and lowered pHi (segment ef). This dissociating intracellular NHd includes not only the NH4f derived from the NH3that entered during segment cd, but also the NH: that directly entered the cell during segment de. Note that the magnitude of the pHi undershoot (compare points c andf) is greater than that of the plateau-phase acidification (compare d and e ) , even though both are caused by the influx of NH:. This apparent discrepancy arises because only a small fraction of the entering NH: dissociates during the plateau phase; the rest dissociates only after removal of external NH3/NH:. We have found that the magnitude of the pHi undershoot upon removal of the external NH,/NH,f is directly related to the amount of NH; that had previously entered. Thus, the small pHi undershoot in Fig. 3A reflects the relatively short exposure to NHJNH:. The importance of the NH: electrochemical gradient is evidenced by an experiment in which this gradient is inverted by depolarizing the axon with high [K+],. Not only does pHi rise during the plateau phase (owing to NH: efflux), but removal of external NH:/NH3 produces a pHi shortfall rather than an undershoot (W. F. Boron and P. De Weer, unpublished observation). I At point d in Fig. 3B, [ N H J ] is ~ approximately equal to [NH,],. If the dissociation constant ( K : ) of the reaction NH; = NH3 + H+is the same inside and outside the cell, then it follows that [H'li INH:li = tG lNH31i = K: [NH31, = [H+I,, [NH:],. In other words, the At point d, EHis -0 m V , equilibrium potential for NH: is the same as that for H + (EH). whereas the membrane potential is ---58 mV. Thus, the electrochemical gradients strongly favor the influx of both H +and NH; . * The fraction of the entering NH: which remains NH: is determined by the relationship between the pK: (-9.5) of the reaction NH: = NH3 + H', and the current pH,. At a pHi of 7.7, the ratio of entering NH; which remains NH: to that which dissociates is about 50 : 1.
REGULATION OF AXONAL pH
255
NH3 is by no means the only weak base that produces sizable increases of intraaxonal pH. Methylamine, triethanolamine, and the K+-channel blocker 4-aminopyridine all cause comparable intracellular alkalinizations (W. F. Boron and P. De Weer, unpublished observation). B. COOand HCO;
When an axon is exposed to a C02-containing solution, pHi falls rapidly (see Fig. 4A, segment ab) as C02 enters the cell and combines with H 2 0 to form H2CO3, which then dissociates into H+ and HCO;. CO2
+ H20 = H2CO3 = H' + HCO;
The entry of C02 and the resultant intraaxonal acidification continue until [C0,li = [CO,],. The extent of the intracellular acidification depends on [CO,],, p, and the initial pHi (because C02 + H 2 0 = H+ + HCO,, the higher [H+],the less the tendency for the reaction to proceed to the right). When the C02/HCO; is removed from the external solution, these processes reverse and pHi returns toward its initial level ( c d ) .However, if, during the C 0 2 exposure, pH, is sufficiently high (e.g., 7 . 9 , pHi returns to a value slightly higher than the initial one (compare points a and d ) . This overshoot is caused by the axon's pHi-regulating system (see below). In the experiment of Fig. 4A, the fall of pHi brought about by the application of C02 appears to be sustained (segment bc) only because the C02 exposure is relatively brief. If the exposure is more prolonged (see Fig. 4B), the initial intracellular acidification (ab) is followed by a slow rise in pHi (bc). This alkalinization cannot be accounted for by passive transport processes, because the predicted passive fluxes of H+ (an influx) or HCO, (an efflux) would each tend to further acidify the axon. The slow segment -bc pH, recovery must therefore be caused by an activetransport mechanism that extrudes acid from the cell (Fig. 4C). When the external C02/HC0; is removed, pHi overshoots its initial value (compare a and d ) by an exaggerated amount. The magnitude of this overshoot is a direct reflection of the amount of acid extruded from the axon during the preceding CO, exposure. Thus the overshoot after the 25-min C02 exposure of Fig. 4A is only about 20% as large as the overshoot following the 125-min C 0 2 exposure of Fig. 4B. Although snail neurons (Thomas, 1976b) and barnacle muscle fibers (Boron et al., 1979) can return pHi to normal (provided pH, and [HCO;], are sufficiently high) after even very large intracellular acid loads (i.e., sufficient to lower pHi by 0.5-1.0), squid axons cannot return pHi to normal during an in vitro experiment unless the acid load is quite small
WALTER F. BORON
5 % co2
B 7.4 75[
'
7-
4
I
>
-
/
50
FIG.4. Effect of Cot on pH,. (A) Brief exposure. An intact axon is exposed to ASW containing 5% c o 2 / 5 0mM HCO;; extracellular pH (pH,) is 7.5 throughout. The influx of C 0 2causes pH, to fall (segment ah) and apparently to level off ( h c ) . The subsequent return to a HCO1-free ASW (buffered with 5 mM Tris-HEPES) produces a slight overshoot of the initial pH, (compare d and a ) . (B) Prolonged exposure. In a more lengthy experiment, it is clear that the initial C02-induced fall in pHi (ah)is followed by a slower recovery ( h c ) .When the COz is now washed out, pH, overshoots its initial value by an exaggerated amount (compare d and ( I ) . (C) Proposed mechanism of segment -6c alkalinization. During the plateau phase of the above C 0 2 exposure, the electrochemical gradients for H' and HCO; favor a net influx and efflux, respectively, of these two ions. Thus, the observed increase in pH, must be due to an active transport process. Although depicted here as a proton extruder, this process could involve the uptake of alkali and/or the efflux of acid. The transport process has been generically described as "acid extrusion." (From Boron and De Weer, 1976a; reproduced by permission of the Rockefeller University Press.)
REGULATION OF AXONAL pH
257
(i.e., lowering pHi by -0. I ) . At a pH, of 7.5, a [HCO.;], of 50 mM, and a pHi of 7.0, the axon’s acid-extrusion rate is exceeding low, being able to raise pHi by only -0.1 pH unit per hour (Boron and De Weer, 1976a). After a few hours, the pH, recovery halts even though pHi is far below its normal value. We suspect that long-term metabolic changes either decrease the acid extrusion rate and/or increase the acid-loading rate, thereby preventing a complete pHi recovery. IV.
EFFECT OF METABOLIC INHIBITORS Off pH1
Cyanide (CN-), 2,4-dinitrophenol (DNP), and azide (Ny) all produce a slow and prolonged intracellular acidification that begins after a short delay. In addition, the latter two substances cause a more rapid but brief phase of acidification that begins very soon after they are applied. As illustrated in Fig. 5A, cyanide has little effect on pHi for the first 5 min, after which it causes pH, to decline by -0.3 over the next -40 min (Boron and De Weer, 1976a). The lack of an immediate acidification implies that the influx of the neutral weak acid (HCN = H+ + CN-) is not followed by a substantial dissociation into H + and CN-. Indeed, the dissociation is expected to be very small because HCN has such a high pK; (-10). The slow fall in pHi is likely the result of a buildup of acidic metabolites,
FIG.5 . (A) Effect of cyanide on pH, and V , . Exposing the axon to ASW containing 2 mM NaCN (pH, = 7.70) causes, after a 5-rnin delay, a slow fall of pH, of -0.3. Simultaneously, there is a transient depolarization, followed by a sustained hyperpolarization. The effect of CN is only partially reversible in the first few minutes. (B) Effect of azide on pH, and V,. Application of 3 mM NaN, (pH, = 7.70) causes a rapid internal acidification followed, after a brief delay, by a slower pH, decline. The membrane voltage undergoes a transient depolarization. followed by a sustained hyperpolarization. The changes are only partially reversible in the first several minutes. (From Boron and De Weer, 1976a; reproduced by permission of the Rockefeller University Press.)
258
WALTER F. BORON
following inhibition of oxidative phosphorylation. It is unlikely to be due to the shuttling of protons across the cell membrane by HCN/CN-, inasmuch as [CN-Ii is exceedingly low (-2 p M ) . Removal of cyanide produces only a small pHi recovery over the first several minutes. The application of azide (3 mM, pH, = 7.70) causes an abrupt fall in pHi of -0.1 (see Fig. SB), followed by a period of stability. If the azide is removed during this period, pHi rapidly returns to its initial value (not shown). Thus, the rapid azide-induced acidification is probably the result of the influx and dissociation of the neutral weak acid (HN3 = Ht + N;; pKA = 4.7). During lengthier exposures to azide, pHi eventually begins a slow decline, similar to the slow phase of acidification produced by cyanide. This slow phase of acidification is likely caused by the build-up of acidic metabolites, the result of azide's uncoupling of oxidative phosphorylation. The inward shuttling of protons across the cell membrane by the HN3/N: couple is probably not a major contributor to this acidification, inasmuch as the slow pHi fall begins only after a lengthy delay, and because reversing the N; electrochemical gradient has only a small effect on pHi (Boron and De Weer, 1976a). Removal of the azide causes a rapid pHi recovery, but only to the same extent as the initial, rapid phase of azide-induced acidification. 2,4-Dinitrophenol (1 mM, pH, = 7.70) has an effect similar to that of azide, except that the slow phase of acidification follows the rapid, initial one without an appreciable delay. V.
IONIC MECHANISM OF pH1 REGULATION
The existence of an axonal pHi-regulating mechanism is indicated by the relatively high value for steady-state pHi (i.e., -7.3 vs the equilibrium pHi of -6.8) and by the plateau-phase recovery of pHi from a C02-induced acid load (see Fig. 4B). These results suggest that an axonal pHi regulator responds to intracellular acid loads by extruding acid from the cell. Detailed mechanistic information can be gained by studying the recovery of pHi from acute, intracellular acid loads. A. Dependence on External HCO;
In the experiment of Fig. 4B, the recovery of pHi from the COJnduced acid load necessarily took place in the presence of external HCO,. The experiment of Fig. 6 was designed to test whether HCO; is required for the recovery of pHi from an acute acid load (Boron and De Weer, 1976b). The acid load is applied by pretreating the axon with an ASW containing 50 mM NH: (pH, = 7.7). As described above, the application of the NH3/
259
REGULATION OF AXONAL pH
F FIG.6 . Effect of external HCO; on recovery of pH, from an acid load. The axon is acid loaded by pretreating it with artificial seawater (ASW) containing 50 mM NH: (pH, = 7.7), and then washing out the NHJNH:. As shown in Fig. 3B, the washout causes pH, to undershoot its initial value; i.e., the cell is acid loaded. In the nominal absence of HCO; (segment AB), pH, fails to recover. During the application of ASW containing 10 mM HCO; at the same pH,, however, pH, recovers more rapidly (segment C D ) . After washout of the CO,/HCO;, pH, no longer recovers (point E ) . (From Boron and De Weer, 1976b; reproduced by permission of Macmillan Journals, Ltd.)
NH: causes an abrupt increase in pHi (due to NH3 influx) followed by a slower fall (due to NH; influx). When the external NH3/NH: is washed out, pHi falls to a value substantially lower than its initial one (i.e., the cell is acid loaded). In the nominal absence of HCO, (segment AB), pHi recovers only very slowly, even though pH, is 8. The application of ASW containing 0.4% C 0 2 and 10 mM HCO; at constant pH, causes a transient acidification (BC) due to the influx of C 0 2 .This is followed, however, by a marked acceleration of the pHi recovery (compare CD and AB). This suggests that external HCO; is required for acid extrusion in the squid axon, The rate of the segment -CD recovery can be used to calculate the rate of acid extrusion (i.e., the equivalent net HCO; influx, JFbo3). This is simply the product of the recovery rate (dpHJdt), intracellular buffering power (p),and axonal volume-to-surface ratio ( p ) . A more extensive series of experiments (Boron and Russell, 1983), similar in design to the experiment of Fig. 6 (i.e., in nondialyzed axons),
260
WALTER
KINETICS OF
External HCO, External Na' Internal CIP
F. BORON
TABLE I AXON'SpH,-RECUt.ATING
THE SQUID
SYSTEMS''
30 21 38
2.3 +- 0.2 77 -t 13 84 f 15
10.6 2 0.4 10.3 t 0.6 19.6 1.2
*
Kinetic parameters obtained from nonlinear, leastsquares curve fit. n is number of data points (measurements of net, equivalent HCO, flux); V:,,, is apparent maximal velocity; KL, is substrate concentration at half VLdx. Standard conditions: [Na'], = 425-437 mM, [HCO;], = 12 mM, pH, = 8.0, [el-], = 100 m M , pH, = 6.7.
has shown that the dependence of acid-extrusion rate on [HCO,], follows Michaelis-Menten kinetics (see Table I). The apparent K , for external HCOT is -2.3 mM (pH, = 8.0, pHi = 6.7, T = 22°C) and the apparent VmaXis 10.6 pmol ~ r n sec-' - ~ (at a physiological [CI -Ii of 70-100 mM). The magnitude of the acid-extrusion rate has been carefully documented (Boron and Russell, 1983) in a series of experiments on internally dialyzed axons. Isotopically measured fluxes of Na+ and C1- (see below) were determined under identical conditions (pHi = 6.7, [ClFIi= 150 mM, pH, = 8.00, [HCO;], = 10 mM, (Na'l, = 425 mM, T = 16°C) for the purpose of determining the stoichiometry of acid extrusion. The approach in the acid-extrusion rate experiments was to dialyze the axon with a lowpH fluid until pHi fell to -6.7, at which time dialysis was halted, thereby returning control of pHi to the axon. After dialysis was halted, pHi failed to recover (i.e., increase) until HCOC was added to the external solution. From thc rate of HCOi-stimulated pHi recovcry, axon diameter, and buffering power,' the acid-extrusion rate (J$io,) was computed. In 15 axons, the mean JnHeEOjwas 7.5 pmol cm12 sec-' (see Table 11). This acid extrusion was completely blocked by the anion-flux inhibitor 4-isothiocyano-4'acetamido -2,2'-disulfonate (SITS), as had previously been demonstrated The non-C02 or intrinsic buffering power (PI)was determined by halting dialysis, blocking the pHi,-regulating systems with SITS, and then exposing the axon to ASW containing 0.2 mM NH; at pH 7.75 (see Boron, 1977; Boron and Russell, 1983). PI, the quotient of the calculated change in [NH;], and the measured pHi change, came to 11.2 mM. The total buffering power (&) is the sum of PI and the COz buffering power (PcOz).The latter is calculated from the relation pco, = 2.3 [HCO, Ii,and comes to I .7 mM under the conditions of these experiments. Thus, PT = 11.2 + 1.7 = 12.9 mM.
REGULATION
OF AXONAL
261
pH
TABLE I1
AXON'S ~H,-RECUI.ATING SYSTEM"
STOICHIOMETRY OF THE SQUID
Ion
Na' CIHCO;
Influx
Efflux
3.4 t 0.4 (13) 0.1 t 0.2 (6) -
0.0 t 0.1 (6) 3.9 t 0.2 (15)
-
Net flux (pmol cm sec-I) +3.4
-3.8
1.5
%
0.6
(15)
Mean kSE, with number of experiments given in parentheses. The HCOj flux was calculated from the rate of pH, recovery and I S the net, equivalent HC03 flux referred to in the text. Standard conditions: temperature = 16°C. [Nail, = 425 mM. [HCO,] = 12 mM, pH, = 8.0, [CI 1, = 150 mM, pH, = 6.5.
for this preparation (Russell and Boron, 1976), as well as the snail neuron (Thomas, 1976a) and barnacle muscle (Boron, 1977). 6. Dependence on External Nai
In 1977, Thomas showed that acid extrusion in snail neurons requires external Na+, an observation confirmed on giant barnacle muscle fibers (Boron et ul., 1979). The experiment of Fig. 7 demonstrates how the recovery of pH, from an NHJNH4f-induced acid load depends on "a+], in the squid axon (Boron and Russell, 1983).After removal of external NHl/ NH;, pH, is allowed to fall and level off (segment ab) in a nominally HC0;-free ASW. Application of 10 mM HCO; ASW containing 425 mM Na+ (bc)causes a rapid recovery of pH, (calculated JFbo3 = 9.7 pmol cm-2 sec-I). The pH, recovery is slowed (cd) by reducing "a+], to 15 mM (JE& = 1.1 pmol cm-2 sec-I), and then accelerated (de) by raising "a+], to 100 mM (J$& = 6.4 pmol cm-2 sec-I). The results of a series of such experiments showed that the dependence of acid-extrusion rate on "a+], follows Michaelis-Menten kinetics, with an apparent K , of 77 mM (pH, = 8.00, pH, = 6.7, temperature = 22°C). Not only does acid extrusion require external Na+, but it is accompanied by a net influx of Na+. Unidirectional Na+ effluxes and influxes (measured with 22Na)were determined in experiments in which axons were dialyzed to a pH, of 6.7 and a [CI-1, of 1.50 m M .The pH,-regulating system-linked Na+ flux was defined as the increase in the Na+ flux pro-
262
WALTER F. BORON klOO N$ PHO 7 7 0
,
12 HCOi
[Notlo:
425
pHo 8.00 IS I00
Fie. 7 . Dependence of acid-extrusion rate on [Na+l0.The axon was acid loaded by pretreating it with artificial seawater (ASW) containing 100 mM NH: (pH, = 7.70). After washout of the NH1/NH:, pH, failed to recover from the acid load (segment ub) until HCO; was added to the ASW. The subsequent pH, recovery was rapid when "a'], was 425 mM ( h c ) , but it slowed considerably when "a+], was reduced to 15 mM ( c d ) . Raising "a+], to 100 mM (de) brought the pH, recovery rate to a value intermediate between the first two values. (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
duced by the application of 10 mM HCO, (pH, = 8.0) ASW. We found that, under standard conditions, the unidirectional Na+ efflux mediated by the axon's pHi-regulating system is approximately zero, and that the influx is about 3.4 pmol cm-2 sec-' (Boron and Russell, 1983). Thus, the pHi-regulating system mediates a net Na' influx (see Table 11). This net Na+ influx shares all the properties of the HCO: flux: (1) it is blocked by SITS; (2) it requires internal C1-; and (3) it requires internal ATP. The
263
REGULATION OF AXONAL pH pH 6.6 k 2 0 0 CI-
Inside:
'
pH, 8.00
'
Outside:
12 HCOj
I
5:"l
7.4
7.2
350 CI-
pHi 7.0
6.8
6.6
30 rnin
FIG.8. Dependence of acid-extrusion rate on [CI-1, . The axon was loaded by predialyzing (segment ab) it with a fluid at pH 6.6, containing 200 mM C1-. When dialysis was halted, returning control of pH, to the axon ( b c ) ,there was no pH, recovery until 12 m M HCO, was added to the artificial seawater (cd). The inset shows the results of three similar experiments (during segments bc and c d ) in which the axons were dialyzed to [CI-1, levels of 0, 100, and 350 mM CI-. (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
magnitude of the net Na+ influx is about 0.45 as large as the J;;CO3obtained under identical conditions. This is consistent with a HCO, : Na+ stoichiometry of 2 : 1. C. Dependence on Internal CI-
When internal CI- is removed by dialyzing the axon with a C1-free solution, acid extrusion is blocked (Russell and Boron, 1976). This dependence on internal CI- is demonstrated by the experiment of Fig. 8 . The axon is dialyzed with a low-pH solution containing 200 mM CIF (segment ab). After dialysis is halted, returning control of pHi to the axons, there is no pHi recovery (bc) until HCOj is added to the external solution. The HCOy-stimulated rise in pHi (at the beginning of the segment cd pHi recovery) corresponds to an acid-extrusion rate of 15.0 pmol cm-2 sec-I. The inset shows three additional experiments (each on a different axon) at [Cl-Ii values of 0, 100, and 350 mM. It is clear that the pHi-recovery rate gradually increases as [ClFIi is raised. The results of 38 similar experi-
264
WALTER
F. BORON
ments show that the dependence of acid-extrusion rate on [C1-Ii follows Michaelis-Menten kinetics, with an apparent K , for internal C1- of 84 mM (see Table I). This value is in the range of reported normal values of [CIVliin squid axons (Brinley and Mullins, 1965) and indicates that the axon's pHi-regulating system is normally only about half saturated with respect to intracellular C1- . This is in marked contrast to the other two substrates (i.e., external Na+ and HCO,), for which the transporter is nearly saturated under physiological conditions. The dependence of acid extrusion on internal C1- has also been demonstrated for snail neurons by Thomas (1977). Acid extrusion not only has an absolute dependence on internal C1-, it is accompanied by a net CIV efflux. When the pHi of dialyzed axon is lowered to 6.7 under the aforementioned standard conditions (pHi = 6.7, [Cl-li = 150 mM, pH, = 8.00, [HCO,], = 10 mM, "a+], = 425 m M , temperature = 16°C) the addition of 10 mM HCO, to the ASW has minimal effect (stimulation of -0. I pmol ern--* sec - I ) on the unidirectional CIinflirx (see Table II), measured with "CI. The CIV c f f h x , however, increases by about 3.9 pmol cm-2 sec-'. The net CI- efflux, 3.8 pmol cm-* sec-I, is 51% as large as the net equivalent HCOY flux, but is approximately the same as the net N a + influx. The external HCOy-stimulated C1efflux has all the properties expected of a flux tightly linked to the pHiregulating system (Boron and Russell, 1983): it ( I ) is blocked by SITS, (2) requires external Na+, (3) requires internal ATP, and (4) is stimulated by IOW pHi. D. Dependence on Internal ATP
If squid axons are treated with either cyanide (2 mM) or DNP (1 mM), pHi fails to recover from a C02-induced acid load. This inhibition is reversible in both cases (Boron and De Weer, 1976b). Although Caldwell (1960) had previously shown that treatment with cyanide or DNP reversibly lowers [ATPI, substantially, the above experiments left open the possibility that cyanide and DNP inhibit the pHi regulator directly, not through their effect on ATP levels. The experiment of Fig. 9 was designed to determine whether, in the continuous presence of cyanide, the addition of ATP would restore the axon's ability to recover from an acid load. In the first part of the experiment, the axon is dialyzed with an ATP-free solution for 1 hr; the dialysate's pH is lowered to 6.6 during the final 30 min to acid load the axon. When dialysis is halted (point a ) , returning control of pH, to the axon, there is no pHi recovery (segment u b ) , even
265
REGULATION OF AXONAL pH INTERNAL FLUID
-
ATP fro. pH Z3
'
pH 6.6
EXTERNAl FLUID
~
8mMATP pH 7.3' pH 6.6
, HC03
FIG.9. Dependence of acid extrusion on ATP. The axon was exposed throughout to artificial seawater containing 2 mM CN. Predialysis with an ATP-free fluid at pH 7.3 was followed with a similar fluid at pH 6.6. After pHi fell to -6.8, dialysis was halted, returning control of pH, to the axon. There was no pH, recovery, however, either in the absence (segments ah and c d ) or in the presence of HCO;. After ATP was reintroduced to the axon (de), in the continued presence of ATP, the application of HCO, elicited a pH, recovery (fg) which was blocked by SITS ( g h ) .(From Russell and Boron, 1976; reproduced by permission of Macmillan Journals. Ltd.)
during stimulation with HCO.7 (bc). After the axon is dialyzed with an ATP-containing solution ( d e ) ,however, application of HCO, does elicit a recovery of pHi from the acid load ( f g ) , and this recovery is blocked by SITS ( g h ) . Thus, ATP or a related substance is required for acid extrusion. As noted above, ATP is also required for the net Na+ influx and C1efflux that are coupled to acid extrusion. Although ATP is necessary for transport, it is not clear whether hydrolysis of ATP is stoichiometrically linked to transport, or whether ATP merely fills a catalytic role. E. Possible Mechanisms of Transport
The above data indicate that the squid axon possesses a SITS-sensitive ion-transport mechanism that responds to intracellular acid loads by extruding acid from the cell. The transport not only requires external HCO? and Na+ and internal C1-, but is accompanied by a net Na+ influx and a net C1- efflux. The data are consistent with a stoichiometry of one Na+ entering the axon for each C1- exiting, and for every two acid equivalents
266
WALTER F. BORON
neutralized inside the axon (see Table 11). The implication of the stoichiometry data that transport is electrically neutral is corroborated by the observation that changes in membrane potential are without significant effect on the acid extrusion rate. These data are accounted for by each of the models of Fig. 10. The first model (Thomas, 1977) has Nat and HCOj entering in exchange for C1- and H+. In the second, the exit of H+ is replaced by the entry of a second HCO,. In the third model, the entry of a single C0:- replaces that of two HCOT. Finally, according to the last model (Becker and Duhrn, 1978; Boron, 1980), Na+ and COi- enter only after forming the ion pair NaCOT (Garrels et al., 1961). These models are equivalent thermodynamically. The free-energy change associated with the overall transport process (AGnec)is the sum of the individual freeenergy changes of the transported ions. In the following example, these free-energy changes are computed for the relatively low pHi of 6.7, assuming normal values for [Cl-]; and “a+];. The calculations are based on the first model of Fig. 10. OUT
IN
FIG.10. Models of acid extrusion. The models are equivalent thermodynamically. They all predict electroneutral transport with a stoichiometry equivalent to one NaCtaken up for each C1- lost, and for each two protons neutralized inside the cell tie., by the uptake of HCO; or a related species and/or the efflux of H+). (From Boron and Russell, 1983; reproduced by permission of the Rockefeller University Press.)
267
REGULATION OF AXONAL pH
AGNa = RT In(INa+],/INa+l,) RT In([HCOi],/[HCOj],) ACcl = RT In([CI-l,/[CI 1,) AGH = RT In([H+],/[H+],)
AGHCo, =
RT ln(50/425) RT ln(0.5/10) = RT ln(550/100) = RT ln(10'8/10-6 7, =
=
=
= = =
-2.14RT -3.00RT +1.70RT -3.00 RT
Thus, the overall free-energy change of the acid-extrusion reaction (pHi = 6.7) is -6.44 RT, the negative value indicating that the reaction ought to proceed spontaneously as written (i.e., extruding acid from the cell). At higher pHi values the calculated AG,,, is less negative (i.e., [H+Iifalls and [HCOjli rises). However, even at the normal pHi 7.3, where the observed is greatly reduced and the accompanying Na+ and C1- fluxes are undetectably low, the AG,,, is still -3.66 RT, and thus still favors the net forward reaction by a wide margin. A similar relationship among AGnet,acid extrusion, and pHi exists in barnacle muscle. In this cell, pHi is regulated by a Na/HCO,-Cl/H exchanger similar to that of the squid axon. The acid-extrusion rate in barnacle muscle falls to near zero at the pHi of 7.3-7.4, even though there is sufficient energy in the ion gradients to drive pHi to -8. The barnacle's pHi-regulating system also mediates bidirectional C1- fluxes and bidirectional Na+ fluxes. Interestingly, all these fluxes are blocked at pHi values greater than -7.3 (Boron et al., 1979; Russell et al., 1983). Thus, all known modes of ion transport by the pHi regulator are blocked at pHi values exceeding 7.4. It is unlikely that the inhibition of all modes of transport at pHi > 7.4 is due solely to unavailability of substrate (i.e., intracellular H+). Indeed, if the apparent transporter-mediated C1- influx and Na+ efflux were due to the microscopic reversibility of the pHi regulator, then one might expect low [H+Iito stimulate rather than inhibit these fluxes. It would thus appear that there is an allosteric site for H+ on the internal surface of the barnacle muscle's pHi regulator. We would predict that this site must be protonated in order for the transporter to operate in any mode. Inasmuch as the squid axon's pHi regulator does not appear to mediate an appreciable Cl- influx or Na+ efflux during acid extrusion (see Sections V,B and V,C), it will be difficult to establish the presence or the absence of a similar allosteric H+ site in this preparation. It is interesting to note that the Na-H exchanger, which regulates pHi in several vertebrate cell types, has a pHi dependence that is similar to that of the Na/ HCO,-Cl/H system (see Aickin and Thomas, 1977; Boron and Boulpaep, 1982a), which suggests that it may have an internal allosteric site for H+. This suspicion has been confirmed by the vesicle studies of Aronson et al. (1982).
268
WALTER F. BORON
Concerning the ATP dependence of the squid axon’s pHi regulator, the preceding thermodynamic calculations point out that there is more than sufficient energy in the gradients of the transported ions to account for the activity of the pH, regulator. If the ion gradients were the only source of energy for the transporter, then one might expect transport to bc reversed by appropriately inverting the gradient of one or more of the transported ions. When such gradient inversions are imposed on the barnacle muscle’s Na/HCO,-CI/H exchanger, for example, transport clearly reverses (Russell et ul., 1983). Reversal of the pH, regulator, however, has yet to be demonstrated in squid axons, despite considerable effort (Boron, unpublished). The squid axon pHi regulator’s dependence on ATP and its resistance to reversal could be explained by a tight coupling of transport to ATP hydrolysis. However, we cannot yet rule out the possibility that ATP is required merely for catalytic purposes, and that reversal of acid extrusion is difficult to observe only for kinetic reasons. Although the four models of Fig. 10 are equivalent thermodynamically, they do not necessarily make the same kinetic predictions. In particular, the ion-pair model predicts that the acid-extrusion rate should not depend on “a’],, or [HCO;], per se, but on [NaCOj] at a given pH. Indeed, the Na+ and HC03- data of Table I indicate that whether [Na ‘I, is varied at a constant [HCOj], of 12 mM or [HCOI], is varied at a constant “a+] of 425 mM, the acid-extrusion rates fall on the same velocity vs [NaCO;], curve. The apparent K , for NaCOr is 74 W M (Boron and Russell, 1983). More recently, these studies have been expanded to include the [Na’], dependence of acid extrusion at three [HCOI] levels (12, 6 , and 3 mM), and the [HCOj], dependence at three “a+] levels (425, 212, and 106 rnM) (Boron, unpublished). In all cases, the data fall on the same velocity vs [NaCOy1, curve, in agreement with the ion-pair model. REFERENCES Aickin, C . C., and Thomas, R. C. (1977). An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers. J . Physiol. (London) 273, 295-3 16. Aronson, P . S . , Nee, J . , and Suhm, M. A. (1982). Modifier role of internal H’ in activating the Na-H exchanger in renal microvillus vesicles. Nature (London) 299, 161-163. Recker, B . F., and Duhm, J. (1978). Evidence for anionic cation transport of lithium, sodium and potassium across the human erythrocyte membrane induced by divalent anions. J . Physio/. (London) 282, 149- 168. Bicher, H . , and Ohki, S. (1972). Intracellular pH electrode experiments on the giant squid axon. Biochirn. Biophys. Aria 255, 900-904. Boron, W. F. (1977). lntracellular pH lransients in giant barnacle muscle fibers. A m . J . Physiol. 233, C61-C73. Boron, W. F. (1980). Intracellular pH regulation. Curr. T ( J ~ Membr. . Trcinsp. 13, 3-22.
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