Biomedical subjects

T E DeCoursey

Publications and source records attributed to T E DeCoursey.

At least 19 recordsLinked to original sources

PubMed2001

Interactions between NADPH oxidase-related proton and electron currents in human eosinophils.

1. Proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, with an applied NH4+ gradient to control pH(i). 2. Voltage-gated proton channels in unstimulated human eosinophils studied with the permeabilized-patch approach had properties similar to those reported in whole-cell studies. 3. Superoxide anion (O2-) release assessed by cytochrome c reduction was compared in human eosinophils and neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated O2 release was more transient and the maximum rate was three times greater in eosinophils. 4. In PMA-activated eosinophils, the H+ current amplitude (I(H)) at +60 mV increased 4.7-fold, activation was 4.0 times faster, deactivation (tail current decay) was 5.4 times slower, the H+ conductance-voltage (g(H)-V) relationship was shifted -43 mV, and diphenylene iodinium (DPI)-inhibitable inward current reflecting electron flow through NADPH oxidase was activated. The data reveal that PMA activates the H+ efflux during the respiratory burst by modulating the properties of H+ channels, not simply as a result of NADPH oxidase activity. 5. The electrophysiological response of eosinophils to PMA resembled that reported in human neutrophils, but PMA activated larger proton and electron currents in eosinophils and the response was more transient. 6. ZnCl2 slowed the activation of H+ currents and shifted the g(H)-V relationship to more positive voltages. These effects occurred at similar ZnCl2 concentrations in eosinophils before and after PMA stimulation. These data are compatible with the existence of a single type of H+ channel in eosinophils that is modulated during the respiratory burst.

Electrons↗

PubMed2001

Activation of NADPH oxidase-related proton and electron currents in human eosinophils by arachidonic acid.

1. Effects of arachidonic acid (AA) on proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, using an applied NH4+ gradient to control pH(i). 2. Superoxide anion (O2-) release was assessed by cytochrome c reduction in human eosinophils. Significant O2- release was stimulated by 5-10 microM AA. 3. AA activated diphenylene iodinium (DPI)-inhibitable inward current reflecting electron efflux through NADPH oxidase. These electron currents (I(e)) were elicited in human eosinophils at AA concentrations (3-10 microM) similar to those that induced O2- release. 4. The voltage-gated proton conductance (g(H)) in eosinophils stimulated with AA was profoundly enhanced: H+ current amplitude (I(H)) increased 4.6 times, activation was 4 times faster, and the H+ conductance-voltage (g(H)-V) relationship was shifted to substantially more negative voltages. The electrophysiological effects of AA resembled those reported for PMA, except that AA did not consistently slow tau(tail) (deactivation of H+ currents). 5. The stimulation of both proton and electron currents by AA was reversible upon washout. Repeated exposure elicited repeated responses. The activation of H+ currents by AA was dissociable from its activation of NADPH oxidase; H+ currents were enhanced at low concentrations of AA that did not elicit detectable I(e) or when NADPH oxidase was inhibited by DPI. 6. Most of the effects of AA on H+ currents qualitatively resemble those reported in whole-cell studies, reflecting a more direct action than PMA. The results are compatible with AA being an immediate activator of both NADPH oxidase and proton channels in human eosinophils.

Arachidonic Acid↗

PubMed2001

The gp91phox component of NADPH oxidase is not the voltage-gated proton channel in phagocytes, but it helps.

During the "respiratory burst," the NADPH oxidase complex of phagocytes produces reactive oxygen species that kill bacteria and other invaders (Babior, B. M. (1999) Blood 93, 1464-1476). Electron efflux through NADPH oxidase is electrogenic (Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) Biochem. J. 246, 325-329) and is compensated by H(+) efflux through proton channels that reportedly are contained within the gp91(phox) subunit of NADPH oxidase. To test whether gp91(phox) functions as a proton channel, we studied H(+) currents in granulocytes from X-linked chronic granulomatous disease patients lacking gp91(phox) (X-CGD), the human myelocytic PLB-985 cell line, PLB-985 cells in which gp91(phox) was knocked out by gene targeting (PLB(KO)), and PLB-985 knockout cells re-transfected with gp91(phox) (PLB(91)). H(+) currents in unstimulated PLB(KO) cells had amplitude and gating kinetics similar to PLB(91) cells. Furthermore, stimulation with the phorbol ester phorbol 12-myristate 13-acetate increased H(+) currents to a similar extent in X-CGD, PLB(KO), and PLB(91) cells. Thus, gp91(phox) is not the proton channel in unstimulated phagocytes and does not directly mediate the increase of proton conductance during the respiratory burst. Changes in H(+) channel gating kinetics during NADPH oxidase activity are likely crucial to the activation of H(+) flux during the respiratory burst.

Carcinogens↗

PubMed2001

Voltage-gated proton channels in microglia.

Microglia, macrophages that reside in the brain, can express at least 12 different ion channels, including voltage-gated proton channels. The properties of H+ currents in microglia are similar to those in other phagocytes. Proton currents are elicited by depolarizing the membrane potential, but activation also depends strongly on both intracellular pH (pH(i)) and extracellular pH (pH(o)). Increasing pH(o) or lowering pH(i) promotes H+ channel opening by shifting the activation threshold to more negative potentials. H+ channels in microglia open only when the pH gradient is outward, so they carry only outward current in the steady state. Time-dependent activation of H+ currents is slow, with a time constant roughly 1 s at room temperature. Microglial H+ currents are inhibited by inorganic polyvalent cations, which reduce H+ current amplitude and shift the voltage dependence of activation to more positive potentials. Cytoskeletal disruptive agents modulate H+ currents in microglia. Cytochalasin D and colchicine decrease the current density and slow the activation of H+ currents. Similar changes of H+ currents, possibly due to cytoskeletal reorganization, occur in microglia during the transformation from ameboid to ramified morphology. Phagocytes, including microglia, undergo a respiratory burst, in which NADPH oxidase releases bactericidal superoxide anions into the phagosome and stoichiometrically releases protons into the cell, tending to depolarize and acidify the cell. H+ currents may help regulate both the membrane potential and pH(i) during the respiratory burst. By compensating for the efflux of electrons and counteracting intracellular acidification, H+ channels help maintain superoxide anion production.

Animals↗

PubMed2000

Simultaneous activation of NADPH oxidase-related proton and electron currents in human neutrophils.

Generation of reactive oxygen species by the NADPH oxidase complex is an important bactericidal weapon of phagocytes. Phorbol myristate acetate (PMA) is a potent agonist for this "respiratory burst" in human neutrophils. Although stoichiometric H(+) efflux occurs during the respiratory burst, efforts to stimulate voltage-gated H(+) channels by PMA in whole-cell patch-clamped phagocytes have been unsuccessful. We have used a modification of the permeabilized-patch configuration that allows control of intracellular pH and preserves second-messenger pathways. Using this method, we show that PMA dramatically enhances and alters voltage-gated proton currents in human neutrophils. PMA produced four alterations in H(+) current properties, each of which increases the H(+) current at any given voltage: (i) a 40-mV negative shift in the H(+) conductance-voltage (g(H)-V) relationship; (ii) faster activation [smaller activation time constant (tau(act))] during depolarizing pulses; (iii) slower deactivation [larger deactivation time constant (tau(tail))] on repolarization; and (iv) a larger maximum H(+) conductance (g(H, max)). Inward current that directly reflects electron transport by NADPH oxidase was also activated by PMA stimulation. The identity of this electron current was confirmed by its sensitivity to diphenylene iodinium, an inhibitor of NADPH oxidase. Diphenylene iodinium also reversed the slowing of tau(tail) with a time course paralleling the inhibition of electron current. However, the amplitudes of H(+) and electron currents activated by PMA were not correlated. A complex interaction between NADPH oxidase and voltage-gated proton channels is indicated. The data suggest that PMA stimulation modulates preexisting H(+) channels rather than inducing a new H(+) channel.

Adult↗

PubMed2000

Common themes and problems of bioenergetics and voltage-gated proton channels.

The existence of a proton-selective pathway through a protein is a common feature of voltage-gated proton channels and a number of molecules that play pivotal roles in bioenergetics. Although the functions and structures of these molecules are quite diverse, the proton conducting pathways share a number of fundamental properties. Conceptual parallels include the translocation by hydrogen-bonded chain mechanisms, problems of supply and demand, equivalence of chemical and electrical proton gradients, proton wells, alternating access sites, pK(a) changes induced by protein conformational change, and heavy metal participation in proton transfer processes. An archetypal mechanism involves input and output proton pathways (hydrogen-bonded chains) joined by a regulatory site that switches the accessibility of the bound proton from one 'channel' to the other, by means of a pK(a) change, molecular movement, or both. Although little is known about the structure of voltage-gated proton channels, they appear to share many of these features. Evidently, nature has devised a limited number of mechanisms to accomplish various design strategies, and these fundamental mechanisms are repeated with minor variation in many superficially disparate molecules.

Cell Membrane↗

PubMed2000

Hypothesis: do voltage-gated H(+) channels in alveolar epithelial cells contribute to CO(2) elimination by the lung?

Although alveolar epithelial cells were the first mammalian cells in which voltage-gated H(+) currents were recorded, no specific function has yet been proposed. Here we consider whether H(+) channels contribute to one of the main functions of the lung: CO(2) elimination. This idea builds on several observations: 1) some cell membranes have low CO(2) permeability, 2) carbonic anhydrase is present in alveolar epithelium and contributes to CO(2) extrusion by facilitating diffusion, 3) the transepithelial potential difference favors selective activation of H(+) channels in apical membranes, and 4) the properties of H(+) channels are ideally suited to the proposed role. H(+) channels open only when the electrochemical gradient for H(+) is outward, imparting directionality to the diffusion process. Unlike previous facilitated diffusion models, HCO(-)(3) and H(+) recombine to form CO(2) in the alveolar subphase. Rough quantitative considerations indicate that the proposed mechanism is plausible and indicate a significant capacity for CO(2) elimination by the lung by this route. Fully activated alveolar H(+) channels extrude acid equivalents at three times the resting rate of CO(2) production.

Acid-Base Equilibrium↗

PubMed1999

pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations.

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl(2) reduced the H(+) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(+) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.

Algorithms↗

PubMed1998

HERG-like K+ channels in microglia.

A voltage-gated K+ conductance resembling that of the human ether-à-go-go-related gene product (HERG) was studied using whole-cell voltage-clamp recording, and found to be the predominant conductance at hyperpolarized potentials in a cell line (MLS-9) derived from primary cultures of rat microglia. Its behavior differed markedly from the classical inward rectifier K+ currents described previously in microglia, but closely resembled HERG currents in cardiac muscle and neuronal tissue. The HERG-like channels opened rapidly on hyperpolarization from 0 mV, and then decayed slowly into an absorbing closed state. The peak K+ conductance-voltage relation was half maximal at -59 mV with a slope factor of 18.6 mV. Availability, assessed by a hyperpolarizing test pulse from different holding potentials, was more steeply voltage dependent, and the midpoint was more positive (-14 vs. -39 mV) when determined by making the holding potential progressively more positive than more negative. The origin of this hysteresis is explored in a companion paper (Pennefather, P.S., W. Zhou, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:795-805). The pharmacological profile of the current differed from classical inward rectifier but closely resembled HERG. Block by Cs+ or Ba2+ occurred only at millimolar concentrations, La3+ blocked with Ki = approximately 40 microM, and the HERG-selective blocker, E-4031, blocked with Ki = 37 nM. Implications of the presence of HERG-like K+ channels for the ontogeny of microglia are discussed.

Animals↗

PubMed1998

Idiosyncratic gating of HERG-like K+ channels in microglia.

A simple kinetic model is presented to explain the gating of a HERG-like voltage-gated K+ conductance described in the accompanying paper (Zhou, W., F.S. Cayabyab, P.S. Pennefather, L.C. Schlichter, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:781-794). The model proposes two kinetically distinct closing pathways, a rapid one favored by depolarization (deactivation) and a slow one favored by hyperpolarization (inactivation). The overlap of these two processes leads to a window current between -50 and +20 mV with a peak at -36 mV of approximately 12% maximal conductance. The near absence of depolarization-activated outward current in microglia, compared with HERG channels expressed in oocytes or cardiac myocytes, can be explained if activation is shifted negatively in microglia. As seen with experimental data, availability predicted by the model was more steeply voltage dependent, and the midpoint more positive when determined by making the holding potential progressively more positive at intervals of 20 s (starting at -120 mV), rather than progressively more negative (starting at 40 mV). In the model, this hysteresis was generated by postulating slow and ultra-slow components of inactivation. The ultra-slow component takes minutes to equilibrate at -40 mV but is steeply voltage dependent, leading to protocol-dependent modulation of the HERG-like current. The data suggest that "deactivation" and "inactivation" are coupled through the open state. This is particularly evident in isotonic Cs+, where a delayed and transient outward current develops on depolarization with a decay time constant more voltage dependent and slower than the deactivation process observed at the same potential after a brief hyperpolarization.

Algorithms↗

PubMed1998

Temperature dependence of voltage-gated H+ currents in human neutrophils, rat alveolar epithelial cells, and mammalian phagocytes.

H+ currents in human neutrophils, rat alveolar epithelial cells, and several mammalian phagocyte cell lines were studied using whole-cell and excised-patch tight-seal voltage clamp techniques at temperatures between 6 and 42 degrees C. Effects of temperature on gating kinetics were distinguished from effects on the H+ current amplitude. The activation and deactivation of H+ currents were both highly temperature sensitive, with a Q10 of 6-9 (activation energy, Ea, approximately 30-38 kcal/mol), greater than for most other ion channels. The similarity of Ea for channel opening and closing suggests that the same step may be rate determining. In addition, when the turn-on of H+ currents with depolarization was fitted by a delay and single exponential, both the delay and the time constant (tauact) had similarly high Q10. These results could be explained if H+ channels were composed of several subunits, each of which undergoes a single rate-determining gating transition. H+ current gating in all mammalian cells studied had similarly strong temperature dependences. The H+ conductance increased markedly with temperature, with Q10 >/= 2 in whole-cell experiments. In excised patches where depletion would affect the measurement less, the Q10 was 2.8 at >20 degrees C and 5.3 at <20 degrees C. This temperature sensitivity is much greater than for most other ion channels and for H+ conduction in aqueous solution, but is in the range reported for H+ transport mechanisms other than channels; e.g., carriers and pumps. Evidently, under the conditions employed, the rate-determining step in H+ permeation occurs not in the diffusional approach but during permeation through the channel itself. The large Ea of permeation intrinsically limits the conductance of this channel, and appears inconsistent with the channel being a water-filled pore. At physiological temperature, H+ channels provide mammalian cells with an enormous capacity for proton extrusion.

Animals↗

PubMed1997

Deuterium isotope effects on permeation and gating of proton channels in rat alveolar epithelium.

The voltage-activated H+ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D2O, for water, H2O, on both the conductance and the pH dependence of gating were explored. D+ was able to permeate proton channels, but with a conductance only about 50% that of H+. The conductance in D2O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D+ than H+), suggesting that D+ interacts specifically with the channel during permeation. Evidently the H+ or D+ current is not diffusion limited, and the H+ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H+ (or D+) and not OH- is the ionic species carrying current. The voltage dependence of H- channel gating characteristically is sensitive to pH0 and pHi and was regulated by pD0 and pDi in an analogous manner. shifting 40 mV/U change in the pD gradient. The time constant of H+ current activation was about three times slower (T(act) was larger) in D2O than in H2O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H+ channel activation requires deprotonation of the channel. In contrast, deactivation (T(tail)) was slowed only by a factor < or = 1.5 in D2O. The results are interpreted within the context of a model for the regulation of H+ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861-896). Most of the kinetic effects of D2O can be explained if the pKa of the external regulatory site is approximately 0.5 pH U higher in D2O.

Animals↗

PubMed1997

Proton and chloride currents in Chinese hamster ovary cells.

Proton and anion conductances were studied in Chinese hamster ovary (CHO) cells using tight-seal whole-cell recording. The pipette solution contained tetramethylammonium, TMA+, methanesulfonate, MeSO3-, highly buffered to pH 5.5. An outwardly rectifying anion conductance was observed in nearly all cells, with a relative permeability of MeSO3- < or = 0.5 that of Cl-. The anion conductance was small immediately after establishing whole-cell configuration, increased rapidly to a maximum at approximately 5 min, and then decreased more slowly to a small value over tens of minutes. A small voltage-activated H+ selective conductance was observed in most cells. H+ currents were studied after the Cl- conductance has subsided. The average H+ current at +40 mV at pHo 7.0 was 1.6 pA/pF. This H+ conductance was activated by membrane depolarization and enhanced at high pHo, and exhibited activation and deactivation kinetics comparable with H+ currents in other mammalian cells.

Animals↗

PubMed1996

Ion channels in human THP-1 monocytes.

The THP-1 human monocytic leukemia cell line is a useful model of macrophage differentiation. Patch clamp methods were used to identify five types of ion channels in undifferentiated THP-1 monocytes. (i) Delayed rectifier K+ current, IDR, was activated by depolarization to potentials positive to -50 mV, inactivated with a time constant of several hundred msec, and recovered from inactivation with a time constant approximately21 sec. IDR was inhibited by 4-aminopyridine (4-AP), tetraethylammonium (TEA+), and potently by charybdotoxin (ChTX). (ii) Ca-activated K+ current (ISK) dominated whole-cell currents in cells studied with 3-10 micron [Ca2+]i. ISK was at most weakly voltage-dependent, with reduced conductance at large positive potentials, and was inhibited by ChTX and weakly by TEA+, Cs+, and Ba2+, but not 4-AP or apamin. Block by Cs+ and Ba2+ was enhanced by hyperpolarization. (iii) Nonselective cation current, Icat, appeared at voltages above +20 mV. Little time-dependence was observed, and a panel of channel blockers was without effect. (iv) Chloride current, ICl, was present early in experiments, but disappeared with time. (v) Voltage-activated H+ selective current is described in detail in a companion paper (DeCoursey & Cherny, 1996. J. Membrane Biol. 152:2). The ion channels in THP-1 cells are compared with channels described in other macrophage-related cells. Profound changes in ion channel expression that occur during differentiation of THP-1 cells are described in a companion paper (DeCoursey et al., 1996. J. Membrane Biol. 152:2).

4-Aminopyridine↗

PubMed1996

Voltage-activated proton currents in human THP-1 monocytes.

Depolarization-activated H+-selective currents were studied using whole-cell and excised-patch voltage clamp methods in human monocytic leukemia THP-1 cells, before and after being induced by phorbol ester to differentiate into macrophage-like cells. The H+ conductance, gH, activated slowly during depolarizing pulses, with a sigmoidal time course. Fitted by a single exponential following a delay, the activation time constant, tauact was roughly 10 sec at threshold potentials, decreasing at more positive potentials. Tail currents upon repolarization decayed mono-exponentially at all potentials. The tail current time constant, tautail, was voltage dependent, decreasing with hyperpolarization from 2-3 sec at 0 mV to approximately 200 msec at -100 mV. Surprisingly, although tauact depended strongly on pHo, tautail was completely independent of pHo. H+ currents were inhibited by Zn2+. Increasing pHo or decreasing pHi shifted the voltage-activation relationship to more negative potentials, tending to activate the gH at any given voltage. Studied in excised, inside-out membrane patches, H+ currents were larger and activated much more rapidly at lower bath pH (i.e., pHi). In THP-1 cells differentiated into macrophages, the H+ current density was reduced by one-half, and tauact was slower by about twofold. The properties of H+ channels in THP-1 cells and in other macrophage-related cells are compared.

Animals↗

PubMed1996

Ion channel expression in PMA-differentiated human THP-1 macrophages.

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K+ currents, IDR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K+ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K+ channel (IIR) and a Ca2+-activated maxi-K channel (IBK). IIR was a classical inward rectifier, conducting large inward currents negative to EK and very small outward currents. IIR was blocked in a voltage-dependent manner by Cs+, Na+, and Ba2+, block increasing with hyperpolarization. Block by Na+ and Ba2+ was time-dependent, whereas Cs+ block was too fast to resolve. Rb+ was sparingly permeant. In cell-attached patches with high [K+] in the pipette, the single IIR channel conductance was approximately 30 pS and no outward current could be detected. IBK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was approximately 200-250 pS and at potentials positive to approximately 100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na+. IBK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca2+]. Macroscopic IBK was blocked by external TEA+ with half block at 0.35 mM. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K+ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed.

Base Sequence↗

PubMed1996

Effects of buffer concentration on voltage-gated H+ currents: does diffusion limit the conductance?

The single-channel proton conductance of the voltage-gated H(+)-selective channel, like that of the F0 component of the H(+)-ATPase, is nearly constant over a wide range of pH encompassing the physiological range. To examine the possible contributions of buffer diffusion and buffer-channel proton transfer reactions to this phenomenon, the effects of buffer concentration on voltage-activated H+ currents were explored in voltage-clamped rat alveolar epithelial cells. Changes in the external buffer concentration ([B]o), evaluated using the whole-cell configuration, had only small effects on H+ currents (IH). Lowering [B]o from 100 to 1 mM did not alter the voltage-activation curve or reversal potential (Vrev) but reduced IH, typically by 10-30%. Changes in internal buffer concentration ([B]i), examined in inside-out patches, usually altered IH more distinctly and subtly changed the kinetics. Overall, the effects of changing buffer concentration were small and subtle. The maximum attenuation of the single-channel H+ current at 1 mM buffer was estimated to be approximately 20% at either mouth of the H+ channel. Therefore, the rate-determining step in H+ permeation is neither deprotonation of buffer at the inner mouth of the channel nor protonation of buffer at the external surface. Evidently the rate of H+ permeation through the channel is itself small enough that diffusion of buffer in bulk solution does not directly limit the conductance significantly.

Animals↗

PubMed1995

Voltage-activated proton currents in membrane patches of rat alveolar epithelial cells.

1. Voltage-activated H(+)-selective currents were studied in cell-attached and excised, inside-out patches of membrane from rat alveolar epithelial cells in primary culture. The pH was varied from 5.5 to 7.5 in the pipette (pHo) and in the bath (pHi). 2. H+ currents in cell-attached patches exhibited activation kinetics and voltage dependence intermediate between their behaviour after excision into pH 6.5 and pH 7.5 solutions, consistent with reported pHi values for alveolar epithelial cells. 3. In inside-out patches, increasing pHo shifted the threshold voltage for activating H+ currents and the relationship between the time constant of activation of H+ currents (tau act) and voltage (V) negatively along the voltage axis by 40-50 mV (unit pH)-1. 4. Decreasing pHi shifted the activation threshold and the tau act-V relationship negatively along the voltage axis by 40-50 mV (unit pH)-1. In addition, at lower pHi the activation of H+ currents upon patch depolarization was markedly faster, with tau act increasing approximately 4- to 5-fold per unit pHi. 5. The limiting slope H+ conductance increased only by a factor of 1.7 per unit pH when pHi was lowered in the range 7.5-5.5 (at constant pHo 7.5). This result suggests that H+ permeation through voltage-activated H+ 'channels' occurs by a mechanism distinct from H+ permeation through water-filled ion channels, in which the conductance might be expected to increase in direct proportion to [H+].

Animals↗