The pH dependence of late sodium current in large sensory neurons.

The effects of altering extracellular pH on late Na+ currents were investigated in large dorsal root ganglion neurons from rats (100-300 g), using patch-clamp techniques. The late current amplitude was steeply dependent upon pH over a range which included normal physiological values: raising the pH from 7.3 to 8.3 approximately doubled the amplitude. Whole-cell late currents 60 ms after depolarization to - 30 mV were blocked with an apparent pKa of 6.96. The pH-dependent changes in current amplitude could not be accounted for by the effects of altered surface charge. In recordings of unitary Na+ currents from outside-out membrane patches, acidification promoted channel opening to a reduced conductance level, near one-half of its maximal value. Acidification to pH < 6.0 also changed the kinetics of the current recruited with the lowest threshold from non-inactivating to inactivating, with the elimination of late openings. We conclude that lowering pH from an initial alkaline or neutral value blocks late Na+ current by reducing the number of contributing channels while also reducing the single channel conductance. The pH dependence of late Na+ current helps to explain clinically relevant changes in neuronal excitability in response to small (i.e. < 1 unit) perturbations in extracellular pH.     

Extracellular pH responses in CA1 and the dentate gyrus during electrical stimulation, seizure discharges, and spreading depression

Since neuronal excitability is sensitive to changes in extracellular pH and there is regional diversity in the changes in extracellular pH during neuronal activity, we examined the activity-dependent extracellular pH changes in the CA1 region and the dentate gyrus. In vivo, in the CA1 region, recurrent epileptiform activity induced by stimulus trains, bicuculline, and kainic acid resulted in biphasic pH shifts, consisting of an initial extracellular alkalinization followed by a slower acidification. In vitro, stimulus trains also evoked biphasic pH shifts in the CA1 region. However, in CA1, seizure activity in vitro induced in the absence of synaptic transmission, by perfusing with 0 Ca(2+)/5 mM K(+) medium, was only associated with extracellular acidification. In the dentate gyrus in vivo, seizure activity induced by stimulation to the angular bundle or by injection of either bicuculline or kainic acid was only associated with extracellular acidification. In vitro, stimulus trains evoked only acidification. In the dentate gyrus in vitro, recurrent epileptiform activity induced in the absence of synaptic transmission by perfusion with 0 Ca(2+)/8 mM K(+) medium was associated with extracellular acidification. To test whether glial cell depolarization plays a role in the regulation of the extracellular pH, slices were perfused with 1 mM barium. Barium increased the amplitude of the initial alkalinization in CA1 and caused the appearance of alkalinization in the dentate gyrus. In both CA1 and the dentate gyrus in vitro, spreading depression was associated with biphasic pH shifts. These results demonstrate that activity-dependent extracellular pH shifts differ between CA1 and dentate gyrus both in vivo and in vitro. The differences in pH fluctuations with neuronal activity might be a marker for the basis of the regional differences in seizure susceptibility between CA1 and the dentate gyrus.     

Effect of Extracellular Potassium Accumulation on Muscle Fiber Conduction Velocity: A Simulation Study

A progressive reduction in muscle fiber conduction velocity is typically observed during fatiguing muscle contraction. Although the exact causes of the conduction velocity decrease have not yet been fully established, increasing evidence suggests that changes in extracellular potassium concentration may be largely responsible. In this study, a mathematical model was developed to examine the effect of extracellular potassium concentration on the muscle fiber action potential and conduction velocity. The model was used to simulate changes in extracellular potassium concentration at a range of temperatures and extracellular potassium accumulation during repetitive stimulation of the muscle fiber at 37 °C. The action potential broadened, and its amplitude and conduction velocity decreased as extracellular potassium concentration increased. The potassium-induced changes in action potential shape and conduction velocity were eliminated when the inward rectifier channels were removed from the model. The results support the hypothesis that accumulation of extracellular potassium ions may be a major contributor to the reduction in muscle fiber conduction velocity and loss of membrane excitability during fatiguing contractions. They additionally suggest that inward rectifier currents play a critical role in potassium-induced membrane depolarization, leading to increased sodium inactivation and resulting in the observed reduction in conduction velocity and membrane excitability.     

Extracellular alkaline-acid pH shifts evoked by iontophoresis of glutamate and aspartate in turtle cerebellum.

The effect of glutamate and aspartate iontophoresis on extracellular pH was investigated in the turtle cerebellum in vitro. Both amino acids produced a rapid alkaline transient, typically followed by a prolonged acidification. These responses could be evoked in all layers of the cerebellum. Transition from bicarbonate to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered media amplified the pH shifts. Similar alkaline-acid transients could be evoked in the molecular layer by electrical stimulation of the parallel fibers or the ipsilateral peduncle, or by superfusion of glutamate or aspartate. However, no alkaline shifts were evoked in the granular layer by either parallel fiber or peduncle stimulation. In contrast, the iontophoretically induced alkaline shifts were largest in the granular layer. Compared with the stimulus-evoked alkalinizations, the iontophoretic alkaline shifts were relatively insensitive to Mn2+ or Cd2+. These data suggest that the activity-dependent alkalinization of brain extracellular space is generated by a bicarbonate-independent mechanism related to excitatory synaptic transmission. The results are consistent with a flux of hydrogen ions through cationic channels, but do not support a direct role for voltage-dependent Ca2+ channels. In view of the sensitivity of ion channels to changes in external pH, and the magnitude of the amino acid-induced pH shifts, these results indicate that extracellular pH could play an important modulatory role in excitatory synaptic transmission.     

Endogenous pH shifts facilitate spreading depression by effect on NMDA receptors

Rapid extracellular alkalinizations accompany normal neuronal activity and have been implicated in the modulation of N-methyl-D-aspartate (NMDA) receptors. Particularly large alkaline transients also occur at the onset of spreading depression (SD). To test whether these endogenous pH shifts can modulate SD, the alkaline shift was amplified using benzolamide, a poorly permeant inhibitor of interstitial carbonic anhydrase. SD was evoked by microinjection of 1.2 M KCl into the CA1 stratum radiatum of rat hippocampal slices and recorded by a proximal double-barreled pH microelectrode and a distal potential electrode. In Ringer solution of pH 7.1 containing picrotoxin (but not at a bath pH of 7.4), addition of 10 microM benzolamide increased the SD alkaline shift from 0.20 +/- 0.07 to 0.38 +/- 0.17 unit pH (means +/- SE). This was correlated with a significant shortening of the latency and an increase in the conduction velocity by 26 +/- 16%. In the presence of the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), benzolamide still amplified the alkaline transient, however, its effect on the SD latency and propagation velocity was abolished. The intrinsic modulation of SD by its alkaline transient may play an important role under focal ischemic conditions by removing the proton block of NMDA receptors where interstitial acidosis would otherwise limit NMDA receptor activity.     

Modulation by extracellular pH of low- and high-voltage-activated calcium currents of rat thalamic relay neurons

The effects of changes in the extracellular pH (pH(o)) on low-voltage- (LVA) and high-voltage- (HVA) activated calcium currents of acutely isolated relay neurons of the ventrobasal thalamic complex (VB) were examined using the whole cell patch-clamp technique. Modest extracellular alkalinization (pH 7.3 to 7.7) reversibly enlarged LVA calcium currents by 18.6 +/- 3.2% (mean +/- SE, n = 6), whereas extracellular acidification (pH 7.3 to 6.9) decreased the current by 24.8 +/- 3.1% (n = 9). Normalized current amplitudes (I/I(7.3)) fitted as a function of pH(o) revealed an apparent pK(a) of 6.9. Both, half-maximal activation voltage and steady-state inactivation were significantly shifted to more negative voltages by 2-4 mV on extracellular alkalinization and to more positive voltages by 2-3 mV on extracellular acidification, respectively. Recovery from inactivation of LVA calcium currents was not significantly affected by changes in pH(o). In contrast, HVA calcium currents were less sensitive to changes in pH(o). Although extracellular alkalinization increased maximal HVA current by 6.0 +/- 2.0% (n = 7) and extracellular acidification decreased it by 11.9 +/- 0.02% (n = 11), both activation and steady-state inactivation were only marginally affected by the moderate changes in pH(o) used in the present study. The results show that calcium currents of thalamic relay neurons exhibit different pH(o) sensitivity. Therefore activity-related extracellular pH transients might selectively modulate certain aspects of the electrogenic behavior of thalamic relay neurons.     

The effect of external pH on the delayed rectifying K+ current in cardiac ventricular myocytes

The effects of extracellular pH (pHe) on the delayed rectifying K+ current iKr in rabbit ventricular myocytes were studied using the whole-cell-clamp technique. Since a variety of results have been reported on the effect of pH on expressed hERG channels, our aim was to investigate the effects of pH on iKr channels in their native environment. iKr is reduced by extracellular acidification and its deactivation is faster. Extracellular acidification results in a marked shift of the steady-state activation curve to more positive potentials, while alkalinization does not produce a significant shift. E1/2= - 11.3 mV, -20.2 mV, -21.4 mV at pHe 6.5, 7.4, 8.5 respectively; the slope factor is 6.2 mV, and is not affected by pHe. Deactivation of iKr is biexponential, with time constants of the order of 0.5 s and 10 s at -50 mV. Both time constants decrease with external acidification. Also the contribution of the fast component to the total amplitude becomes larger with acidification. Acidification also decreases the fully activated iKr current. Our experiments demonstrate that extracellular acidification reduces iKr by increasing the rate of deactivation, causing a shift of the voltage dependence of activation and producing a voltage-dependent block of the fully activated iKr current.     

Kinetics of activity-evoked pH transients and extracellular pH buffering in rat hippocampal slices

The kinetics of activity-dependent, extracellular alkaline transients, and the buffering of extracellular pH (pH(e)), were studied in rat hippocampal slices using a fluorescein-dextran probe. Orthodromic stimuli generated alkaline transients < or = 0.05 pH units that peaked in 273 +/- 26 ms and decayed with a half-time of 508 +/- 43 ms. Inhibition of extracellular carbonic anhydrase (ECA) with benzolamide increased the rate of rise by 25%, doubled peak amplitude, and prolonged the decay three- to fourfold. The slow decay in benzolamide allowed marked temporal summation, resulting in a severalfold increase in amplitude during long stimulus trains. Addition of exogenous carbonic anhydrase reduced the rate of rise, halved the peak amplitude, but had no effect on the normalized decay. A simulation of extracellular buffering kinetics generated recoveries from a base load consistent with the observed decay of the alkaline transient in the presence of benzolamide. Under control conditions, the model approximated the observed decays with an acceleration of the CO2 hydration-dehydration reactions by a factor of 2.5. These data suggest low endogenous ECA activity, insufficient to maintain equilibrium during the alkaline transients. Disequilibrium implies a time-dependent buffering capacity, with a CO2/HCO3- contribution that is small shortly after a base load. It is suggested that within 100 ms, extracellular buffering capacity is about 1% of the value at equilibrium and is provided mainly by phosphate. Accordingly, in the time frame of synaptic transmission, small base loads would generate relatively large changes in interstitial pH.     

Stimulus-induced extracellular pH transients in the in vitro turtle cerebellum

In a number of CNS preparations, neuronal activation has been shown to result in a rapid extracellular alkaline transient, followed by a prolonged acid shift. The isolated turtle cerebellum was used to investigate the early alkaline transient. Double-barreled ion-sensitive microelectrodes for H+, K+ and tetramethylammonium were used to measure field potentials and extracellular ion and volume shifts in response to bipolar electrical stimulation of the parallel fibers. Transition from 15 mM HEPES to 35 mM HCO3- -buffered Ringer decreased the amplitude of the alkaline shift, presumably due to a marked increase in extracellular buffering power. In HEPES-buffered Ringer, repetitive stimulation produced alkaline shifts as large as 0.3-0.4 pH. Single shocks produced an alkaline shift of 0.006 +/- 0.0002 pH with a latency as short as 70 ms. Kynurenic acid (an excitatory amino acid antagonist), or Mn2+, blocked the alkaline shift and the postsynaptic component of the field potential. The alkaline shift was not blocked by the Na-H exchange inhibitor amiloride. The relationship between pHo and extracellular volume transients was studied using tetramethylammonium as an extracellular volume indicator. In nominally HCO3-free Ringer, stimulation at 5 Hz for 10 s caused a decrease in extracellular volume of 3.0 +/- 0.2 per cent. The volume transient was unaffected by 3 mM Mn2+, while the alkaline shift was completely abolished. The data for the alkaline shift are consistent with a channel-mediated transmembrane flux of proton equivalents. The size of the pH change and the underlying perturbation it represents, indicate that acid-base shifts may be a functionally important consequence of neuronal activity.     

Excitatory amino acids and intracellular pH in motoneurons of the isolated frog spinal cord

Double-barrelled pH-sensitive micro-electrodes were used to measure changes of intracellular and extracellular pH in and around motoneurons of the isolated frog spinal cord during application of excitatory amino acids. It was found that N-methyl-D-aspartate, quisqualate and kainate produced a concentration-dependent intracellular acidification. Extracellularly, triphasic pH changes (acid-alkaline-acid going pH transients) were observed during the action of these amino acids. The possible significance of such pH changes for the physiological and pathophysiological effects of excitatory amino acids are discussed.     

Extracellular pH dynamics of retinal horizontal cells examined using electrochemical and fluorometric methods

Extracellular H(+) has been hypothesized to mediate feedback inhibition from horizontal cells onto vertebrate photoreceptors. According to this hypothesis, depolarization of horizontal cells should induce extracellular acidification adjacent to the cell membrane. Experiments testing this hypothesis have produced conflicting results. Studies examining carp and goldfish horizontal cells loaded with the pH-sensitive dye 5-hexadecanoylaminofluorescein (HAF) reported an extracellular acidification on depolarization by glutamate or potassium. However, investigations using H(+)-selective microelectrodes report an extracellular alkalinization on depolarization of skate and catfish horizontal cells. These studies differed in the species and extracellular pH buffer used and the presence or absence of cobalt. We used both techniques to examine H(+) changes from isolated catfish horizontal cells under identical experimental conditions (1 mM HEPES, no cobalt). HAF fluorescence indicated an acidification response to high extracellular potassium or glutamate. However, a clear extracellular alkalinization was found using H(+)-selective microelectrodes under the same conditions. Confocal microscopy revealed that HAF was not localized exclusively to the extracellular surface, but rather was detected throughout the intracellular compartment. A high degree of colocalization between HAF and the mitochondrion-specific dye MitoTracker was observed. When HAF fluorescence was monitored from optical sections from the center of a cell, glutamate produced an intracellular acidification. These results are consistent with a model in which depolarization allows calcium influx, followed by activation of a Ca(2+)/H(+) plasma membrane ATPase. Our results suggest that HAF is reporting intracellular pH changes and that depolarization of horizontal cells induces an extracellular alkalinization, which may relieve H(+)-mediated inhibition of photoreceptor synaptic transmission.     

Chemical cross-linking of proteins of the influenza virion

Purified influenza virus (A/FPV/Rostock/34;H7N1) was exposed briefly to pH 5 before returning to an alkaline pH. Virus was then reacted with one of three chemical cross-linking reagents [dimethyl suberimidate (DMS), tartryl diazide (TDA), or formaldehyde which span 11, 6, and 2A, respectively]. Cross-linked polypeptides were analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and identified with monospecific antisera against HA1, HA2, NP and M1. Acidification resulted in changes in the cross-linking patterns for both HA1 and HA2 which could be detected with all three reagents. Most notable were the data with formaldehyde: under alkaline conditions cross-linking gave only HA1:HA2 heteropolymers but after brief acidification none of these were formed and in their place was a novel HA1 homodimer, an HA2 homotrimer and an HA2 of Mr 50k cross-linked to form a homodimer with another HA2 or to a heterodimer with M1. Although cross-linking by formaldehyde was much more affected by acidification of the virus than cross-linking by DMS or TDA, over half the polymers cross-linked by DMS were no longer formed after acidification. The patterns of cross-linking of NP and M1 were unchanged by low pH treatment.     

Phosphate transfer and tubular pH during renal stopped flow microperfusion experiments in the rat

Summary The loss of³²P-phosphate salts by the luminal compartment of cortical tubules was studied in control and in acetazolamide-infused rats, during stopped-flow microperfusion with 100 mM phosphateraffinose solutions. When the initial pH of the perfusion solution was low (5.5), phosphate was lost more rapidly from proximal tubules than at high initial pH (8.2). The average half-time of phosphate loss was 31.9 s during acid, and 66.0 s during alkaline perfusion in proximal tubules of control rats; in acetazolamide-infused rats half-times were 77.0 and 86.6 s for acid and alkaline perfusions. Thus acetazolamide infusion slows the rate of phosphate loss by proximal tubules, when the perfusion solution is acid, but has no significant effect if its pH is alkaline. These half-times compare to proximal acidification rates of 7.43 s in control and 13.2 s in acetazolamide-infused rats. In distal tubules of control rats no significant loss of phosphate was observed during the period of perfusion. It is concluded that the loss of phosphate, in proximal tubules, is markedly slower than the changes in tubular pH and so its effect on tubular acidification must be of minor importance. In distal tubules changes in pH are not due to transepithelial phosphate movement.Supported by Fund. de Amparo à Pesquisa do Estado de São Paulo     

Modulation of spreading depression by changes in extracellular pH

Spreading depression (SD) and related phenomena have been implicated in hypoxic-ischemic injury. In such settings, SD occurs in the presence of marked extracellular acidosis. SD itself can also generate changes in extracellular pH (pH(o)), including a pronounced early alkaline shift. In a hippocampal slice model, we investigated the effect of interstitial acidosis on the generation and propagation of SD in the CA1 stratum radiatum. In addition, a carbonic anhydrase inhibitor (benzolamide) was used to decrease buffering of the alkaline shift to investigate its role in the modulation of SD. pH(o) was lowered by a decrease in saline HCO(3)(-) (from 26 to 13 to 6.5 mM at 5% CO(2)), or by an increase in the CO(2) content (from 5 to 15% in 26 mM HCO(3)(-)). Recordings with pH microelectrodes revealed respective pHo values of 7.23 +/- 0. 13, 6.95 +/- 0.10, 6.67 +/- 0.09, and 6.97 +/- 0.12. The overall effect of acidosis was an increase in the threshold for SD induction, a decrease in velocity, and a shortened SD duration. This inhibition was most pronounced at the lowest pH(o) (in 6.5 mM HCO(3)(-)) where SD was often blocked. The effects of acidosis were reversible on return to control saline. Benzolamide (10 microM) caused an approximate doubling of the early alkaline shift to an amplitude of 0.3-0.4 U pH. The amplified alkalosis was associated with an increased duration and/or increased velocity of the wave. These effects were most pronounced in acidic media (13 mM HCO(3)(-)/5% CO(2)) where benzolamide increased the SD duration by 55 +/- 32%. The initial velocity (including time for induction) and propagation velocity (measured between distal electrodes) were enhanced by 35 +/- 25 and 26 +/- 16%, respectively. Measurements of [Ca(2+)](o) demonstrated an increase in duration of the Ca(2+) transient when the alkaline shift was amplified by benzolamide. The augmentation of SD caused by benzolamide was blocked in media containing the N-methyl-D-aspartate (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid. These data indicate that the induction and propagation of SD is inhibited by a fall in baseline pH characteristic of ischemic conditions and that the early alkaline shift can remove this inhibition by relieving the proton block on NMDA receptors. Under ischemic conditions, the intrinsic alkalosis may therefore enable SD and thereby contribute to NMDA receptor-mediated injury.     

The pH sensitivity of the chloride conductance of frog skeletal muscle

1. The effect of changes in the pH of the extracellular solution on the membrane conductance of frog sartorius and toe muscle fibres was measured with intracellular micro-electrodes.2. In Ringer solution the membrane conductance was found to be highly sensitive to changes in pH between 5.0 and 9.8. In alkaline solution the conductance rose; in acid solution it fell.3. After replacement of chloride by the relatively impermeant methylsulphate ion the membrane conductance showed little change when pH was altered. It is concluded that chloride is the ion species principally concerned in the pH sensitivity of the resting membrane conductance.4. The relation between pH and the chloride conductance was sigmoid, with the steepest part of the curve lying in the region of neutrality.5. The membrane conductance of muscles equilibrated in a 100 mM-K 216 mM-Cl solution was also sensitive to changes of extracellular pH. As in Ringer solution, the membrane conductance rose in alkaline and fell in acid solutions in a sigmoid fashion.6. Sartorius muscles in isotonic potassium methylsulphate solution showed no change in membrane conductance at different pH values.7. In chloride-free solution a fall in pH tended to cause depolarization; a rise in pH had the opposite effect.8. In Ringer solution the initial effect of a rise in pH was usually a transient depolarization. The indication is that the intracellular concentration of chloride ions may be slightly in excess of that which corresponds to the resting potential. The long-term effects of changes in pH on the membrane potential in Ringer solution were in the same direction as in the absence of chloride.9. The transient potential changes produced on addition and withdrawal of chloride ions were found to be larger in alkaline solutions than in acid solutions. This is further evidence for a higher chloride permeability in alkaline solutions.     

Peritubular pH and PCO'2 in renal tubular acidification.

The influence of peritubular capillary pH and PCO'2 on renal tubular acidification was studied in rats by luminal and peritubular perfusion techniques. Luminal stopped-flow microperfusions were carried out with bicarbonate or alkaline phosphate solutions and luminal pH continuously measured by antimony micorelectrodes. Peritubular calpillary microperfusions were carried out with mammalian Ringer solution kept at different pH and PCO'2. The acidification process was assessed in terms of 1)maximal pH differences, 2)rates of pH change, and 3)rates of bicarbinate reabsorption or H'+ ion secretion. During peritubular perfusions at physiological pH and PCO'2 tubular acidifying capacity was maintained at near-normal levels. Perfusingcapillaries at high pH and low PCO'2, especially with bicarbonate Ringer, acidification was markedly depressed; it was moderately reduced at a peritubular pH of 5.6. At a capillary pH of 7.4, acidification was similiar at low and physiological PCO'2and enhanced at elevated PCO'2. Systemic respiratory acidosis enhanced acidification in the proximal tubule, but reduced it in distal segments.     

pH modulation of an inward rectifier chloride current in cultured rat cortical astrocytes

The effects of changes in extra- and intracellular pH in the pathophysiological range (6.0-8.0) on astroglial plasma membrane ionic currents were investigated with the whole-cell patch-clamp technique. In cultured rat neocortical type-1 astrocytes differentiated by a long-term treatment with dibutyryl cyclic-AMP, exposure to an extracellular pH of 6.4 induced, as compared with the control extracellular pH at 7.3, a sustained and reversible increase in the holding current at -60mV. The rise in current was accompanied by a decrease in the apparent input resistance. Ion substitution experiments indicated that extracellular pH 6.4 upregulated the resting Cl(-) conductance, whereas an opposite effect could be observed at extracellular pH 8.0. Recordings of isolated Cl(-) currents showed that this modulation occurred on the previously identified hyperpolarization-activated, inwardly rectifying Cl(-) current, I(Clh). Extracellular acidification to pH 6.4 shifted the voltage dependence of I(Clh) activation by approximately 20mV towards more positive potentials, whereas a approximately 20mV opposite shift was observed upon exposure to extracellular pH 8.0. These effects were paralleled by an increase (extracellular pH 6.4) or decrease (extracellular pH 8.0) in the maximal conductance. Decreasing (6.0) or increasing (8.0) the intracellular pH shifted the steady-state activation of I(Clh) towards more negative or positive potentials, respectively, leaving unchanged the current sensitivity to extracellular pH modifications.The modulation of the inward rectifier Cl(-) current expressed by differentiated cultured neocortical astrocytes indicates that extra- and intracellular changes in pH occurring in a pathophysiological range may contribute to regulating Cl(-) accumulation in astroglial cells.     

Modulation of volume-regulated anion channels by extra- and intracellular pH

 We have studied the modulation of the volume-regulated anion channel (VRAC) in cultured endothelial cells from bovine pulmonary artery (CPAE cells) by extra- and intracellular pH. The patch-clamp technique was used in combination with a fluorimetric measurement of intracellular pH using BCECF. Swelling of CPAE cells was accompanied by a slow acidification. The metabolites lactate and HCO₃ ^– both permeate through VRAC. The inactivation of VRAC currents at positive potentials is accelerated at a decreased extracellular pH and decelerated at alkaline pH. The instantaneous current amplitude is only slightly affected. Intracellular alkalization reduced whereas acidification enhanced the currents flowing through VRAC at all potentials. HCO₃ ^– and lactate permeation, as well as the up-regulation of VRAC at an acidic intracellular pH might be related to a possible role of this channel in cellular pH regulation. Received: 6 April 1998 / Received after revision: 11 May 1998 / Accepted: 12 May 1998     

Activity-dependent changes in extracellular potassium and excitability in turtle olfactory nerve

The excitability properties of turtle olfactory nerve (o.n.) were studied in vitro using potassium-sensitive microelectrodes (KSM), a modified sucrose gap chamber, and a standard nerve chamber to measure conduction velocity. A pronounced supernormal period (SNP), as indicated by increased conduction velocity of the o.n. fiber volley, lasting up to several seconds, was observed following a single stimulus. The compound action potential recorded in the sucrose gap chamber showed a prolonged depolarization with a similar time course to the SNP. When stimulation intensity was submaximal the response amplitude, and the extracellular potassium concentration [K+]o, continuously increased during repetitive stimulation. In contrast, when supramaximal stimuli were applied, the amplitude of the o.n. fiber volley was reduced during a high-frequency stimulus train for all responses after the initial one even though latency was maximally reduced, i.e., during supernormal conduction. Superfusion with various levels of K+ elicited changes in the excitability of the o.n. fibers. Small increases in [K+]o above the resting concentration of 2.6 mM led to an increase in resting excitability, whereas larger increases resulted in decreased excitability and conduction block. The SNP was eliminated when extracellular potassium was elevated between 3 and 4 mM above resting levels. Microstimulation of a small bundle of o.n. fibers led to an increase in [K+]o along the bundle but also around adjacent nonactivated fibers. The excitability of these neighboring nonactivated fibers was increased, further indicating the importance of activity-dependent changes in [K+]o in modulating axonal excitability. These results demonstrate the importance of activity-dependent increases in extracellular potassium in modulating nonmyelinated o.n. fiber excitability. They also indicate that increases in [K+]o and an associated membrane depolarization contribute to the increased excitability observed during fiber recruitment and the supernormal period.     

Modulation of the volume-sensitive K+ current in Ehrlich ascites tumour cells by pH

The effects of extracellular and intracellular pH (pHo and pHi respectively) on the regulatory volume decrease (RVD) response and on the volume-sensitive K+ and Cl- currents (IK,vol and ICl,vol respectively) were studied in Ehrlich ascites tumour cells. Alkaline pHo accelerated and acidic pHo decelerated the RVD response significantly. Intra- and extracellular alkalinisation increased the amplitude of IK,vol whereas acidification had an inhibitory effect. The magnitude of ICl,vol was not affected by changes in pHi or pHo. A significant reduction in the activation time for IK,vol after hypotonic cell swelling was observed upon moderate intracellular alkalinisation (to pHi 7.9). A further increase in pHi to 8.4 resulted in the spontaneous activation of an IK under isotonic conditions which resembled IK,vol with respect to its pharmacological profile and current/voltage (I/V) relation. Noise analysis demonstrated that the increased amplitude of IK,vol at alkaline pH resulted mainly from an increase in the number of channels (N) contributing to the current. The channel open probability, Po, was largely unaffected by pH. The pH dependence and the biophysical and pharmacological properties of IK,vol are similar to those of the cloned tandem pore-domain acid-sensitive K+ (TASK) channels, and in the current study the presence of TASK-1 was confirmed in Ehrlich cells.