K+ channels activated by L-alanine transport in isolated Necturus enterocytes

Using the patch-clamp technique, we demonstrate here the opening of K+ channels evoked by the actively transported amino acid L-alanine in isolated Necturus enterocytes. These channels had a conductance of about 30 pS and their activation was dependent on transmembrane electrical potential and cytosolic Ca2+.     

Modulation of K+ channels by hydrogen peroxide.

External application of hydrogen peroxide (H2O2) was found to inhibit the time-dependent fast inactivation process of three cloned voltage-gated K+ channels expressed in Xenopus oocytes: KShIIIC, KShIIID and HukII. As expected from kinetic models where some channels are still opening while a significant fraction of channels is already inactivated there was a large increase in current magnitude concomitant to inactivation block. The channels otherwise functioned normally. The effects of H2O2 were specific (other cloned voltage-gated K+ channels were not affected), and reversible, the currents returned to normal upon removal of the H2O2. H2O2 is produced during normal metabolism; it could act as a modulator of excitability through effects on K+ channels if effective local concentrations are reached in neuronal regions close to the channel. KShIIIC and KShIIID currents are very similar to an O2-sensitive K+ current present in type I cells of the carotid body which is believed to underlie the modulation of excitability of these cells by changes in arterial O2 pressure. H2O2 has been proposed as an intermediary between O2 and cellular response in the carotid body; our results provide support for this model.     

Gating of inward-rectifier K+ channels by intracellular pH.

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

Modulation of K+ channels by arachidonic acid in T84 cells. I.Inhibition of the Ca2+-dependent K+ channel

TheCl secretory response ofcolonic cells to Ca²⁺-mediatedagonists is transient despite a sustained elevation of intracellular Ca²⁺. We evaluated the effects ofsecond messengers proposed to limit Ca²⁺-mediatedCl secretion on thebasolateral membrane,Ca²⁺-dependentK⁺ channel(K_Ca) in colonic secretorycells, T84. Neither protein kinase C (PKC) nor inositoltetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affectedK_Ca in excised inside-out patches.In contrast, arachidonic acid (AA; 3 µM) potently inhibitedK_Ca, reducingNP_o, the productof number of channels and channel open probability, by 95%. Theapparent inhibition constant for this AA effect was 425 nM. AAinhibited K_Ca in the presence ofboth indomethacin and nordihydroguaiaretic acid, blockers of thecyclooxygenase and lipoxygenase pathways. In the presence of albumin,the effect of AA on K_Ca wasreversed. A similar effect of AA was observed onK_Ca during outside-out recording.We determined also the effect of thecis-unsaturated fatty acid linoleate,the trans-unsaturated fatty acidelaidate, and the saturated fatty acid myristate. At 3 µM, all ofthese fatty acids inhibited K_Ca,reducing NP_o by 72-86%. Finally, the effect of the cytosolic phospholipaseA₂ inhibitorarachidonyltrifluoromethyl ketone(AACOCF₃) on thecarbachol-induced short-circuit current(I_sc) responsewas determined. In the presence ofAACOCF₃, the peakcarbachol-inducedI_sc response wasincreased ~2.5-fold. Our results suggest that AA generation inducedby Ca²⁺-mediated agonists maycontribute to the dissociation observed between the rise inintracellular Ca²⁺ evoked by theseagonists and the associatedCl secretory response.

     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

Ca2+-activated K+ channels in murine endothelial cells: block by intracellular calcium and magnesium

The intermediate (IK(Ca)) and small (SK(Ca)) conductance Ca(2+)-sensitive K(+) channels in endothelial cells (ECs) modulate vascular diameter through regulation of EC membrane potential. However, contribution of IK(Ca) and SK(Ca) channels to membrane current and potential in native endothelial cells remains unclear. In freshly isolated endothelial cells from mouse aorta dialyzed with 3 microM free [Ca(2+)](i) and 1 mM free [Mg(2+)](i), membrane currents reversed at the potassium equilibrium potential and exhibited an inward rectification at positive membrane potentials. Blockers of large-conductance, Ca(2+)-sensitive potassium (BK(Ca)) and strong inward rectifier potassium (K(ir)) channels did not affect the membrane current. However, blockers of IK(Ca) channels, charybdotoxin (ChTX), and of SK(Ca) channels, apamin (Ap), significantly reduced the whole-cell current. Although IK(Ca) and SK(Ca) channels are intrinsically voltage independent, ChTX- and Ap-sensitive currents decreased steeply with membrane potential depolarization. Removal of intracellular Mg(2+) significantly increased these currents. Moreover, concomitant reduction of the [Ca(2+)](i) to 1 microM caused an additional increase in ChTX- and Ap-sensitive currents so that the currents exhibited theoretical outward rectification. Block of IK(Ca) and SK(Ca) channels caused a significant endothelial membrane potential depolarization (approximately 11 mV) and decrease in [Ca(2+)](i) in mesenteric arteries in the absence of an agonist. These results indicate that [Ca(2+)](i) can both activate and block IK(Ca) and SK(Ca) channels in endothelial cells, and that these channels regulate the resting membrane potential and intracellular calcium in native endothelium.     

Modulation of K+ conductance by intracellular pH in pancreatic beta-cells

Measurements of the effects of NH3/NH4+ on glucose-induced electrical activity in beta-cells from microdissected mouse islets of Langerhans and on intracellular pH in single collagenase-isolated islets pre-loaded with a fluorescent pH probe were performed and are reported here. Application of NH3/NH4+ (15 mM) in the presence of glucose (11 mM) promptly hyperpolarized the beta-cell membrane, reduced input resistance by 60% and blocked electrical activity. These changes were paralleled by an increase in islet fluorescence indicative of a cytosolic pH increase. Removal of NH4Cl initially stimulated electrical activity, which returned to resting level with a time constant of 51 s. Concomitant with the removal of NH4Cl there was a drop in pHi followed by a slow return to resting level with a time constant of 83 s. The results suggest that the [Ca2+]-dependent K+ channel in the beta-cell membrane is activated by a rise in cytosolic pH.     

Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pH(i)) and Na(+) activity ([Na(+)](i)) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pH(o)). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pH(o) to 2.0 or 10.3. At constant pH(o), buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO(3)(-)/CO(2) inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO(3)(-)/CO(2) buffer, suggesting a regulatory role for pH(i). In polarized TRCs step changes in apical pH(o) from 10.3 to 2.0 induced a linear decrease in pH(i) that remained within the physiological range (slope = 0.035; r(2) = 0.98). At constant pH(o), perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO(3)(-)/CO(2) decreased resting TRC pH(i), and MK-507 or MK-417 attenuated the decrease in pH(i) in TRCs perfused with HCO(3)(-)/CO(2) buffer. In parallel experiments, TRC [Na(+)](i) decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na(+), and (d) acid loading the cells with NH(4)Cl or sodium acetate at constant pH(o). Diethylpyrocarbonate and Zn(2+), modification reagents for histidine residues in proteins, attenuated the CO(2)-induced inhibition of NaCl CT responses and the pH(i)-induced inhibition of apical Na(+) influx in TRCs. We conclude that TRC pH(i) regulates Na(+)-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.     

Modulation of Ca2+- and voltage-activated K+ channels by internal Mg2+ in salivary acinar cells

High-conductance K+ channels are known to be activated by internal Ca2+ and membrane depolarization. The effects of changes in internal Mg2+ concentration have now been investigated in patch-clamp single-channel current experiments on excised membrane fragments from mouse acinar cells. It is shown that Mg2+ in the concentration range 10(-6)-10(-3) M evokes a dose-dependent K+ channel activation at a constant Ca2+ concentration of 10(-8) M. The demonstration that changes in [Mg2+]i between 2.5 X 10(-4) and 1.13 X 10(-3) M has effects on the channel open-state probability indicates that fluctuations in [Mg2+]i in intact cells may influence the control of channel opening.     

Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.     

Apical membrane Na+/H+ exchange in Necturus gallbladder epithelium. Its dependence on extracellular and intracellular pH and on external Na+ concentration

Intracellular microelectrode techniques and extracellular pH measurements were used to study the dependence of apical Na+/H+ exchange on mucosal and intracellular pH and on mucosal solution Na+ concentration ([Na+]o). When mucosal solution pH (pHo) was decreased in gallbladders bathed in Na(+)-containing solutions, aNai fell. The effect of pHo is consistent with titration of a single site with an apparent pK of 6.29. In Na(+)-depleted tissues, increasing [Na+]o from 0 to values ranging from 2.5 to 110 mM increased aNai; the relationship was well described by Michaelis-Menten kinetics. The apparent Km was 15 mM at pHo 7.5 and increased to 134 mM at pHo 6.5, without change in Vmax. In Na(+)-depleted gallbladders, elevating [Na+]o from 0 to 25 mM increased aNai and pHi and caused acidification of a poorly buffered mucosal solution upon stopping the superfusion; lowering pHo inhibited both apical Na+ entry and mucosal solution acidification. Both effects can be ascribed to titration of a single site; the apparent pK's were 7.2 and 7.4, respectively. Diethylpyrocarbonate (DEPC), a histidine-specific reagent, reduced mucosal acidification by 58 +/- 4 or 39 +/- 6% when exposure to the drug was at pHo 7.5 or 6.5, respectively. Amiloride (1 mM) did not protect against the DEPC inhibition, but reduced both apical Na+ entry and mucosal acidification by 63 +/- 5 and 65 +/- 9%, respectively. In the Na(+)-depleted tissues mean pHi was 6.7. Cells were alkalinized by exposure to mucosal solutions containing high concentrations of nicotine or methylamine. Estimates of apical Na+ entry at varying pHi, upon increasing [Na+]o from 0 to 25 mM, indicate that Na+/H+ exchange is active at pHi 7.4. Intracellular H+ stimulated apical Na+ entry by titration of more than one site (apparent pK 7.1, Hill coefficient 1.7). The results suggest that external Na+ and H+ interact with one site of the Na+/H+ exchanger and that cytoplasmic H+ acts on at least two sites. The external titratable group seems to be an imidazolium, which is apparently different from the amiloride-binding site. The dependence of Na+ entry on pHi supports the notion that the Na+/H+ exchanger is operational under normal transport conditions.     

Modulation of vertebrate brain Na+ and K+ channels by subtypes of protein kinase C.

Effects of purified subtypes I, II and III of protein kinase C (PKC) on voltage-dependent transient K+ (A) and Na+ channels were studied in Xenopus oocytes injected with chick brain RNA. The experiments were performed in the constant presence of 10 nM beta-phorbol 12-myristate-13-acetate (PMA). Intracellular injection of subtype I (tau) reduced the A-current (IA), with no effect on Na+ current (INa). PKC subtype II (beta 1 + beta 2) and III (alpha) reduced both currents. PKC did not affect the response to kainate. Inactivated (heated) or unactivated (injected in the absence of PMA) enzyme and vehicle alone had no effect. Our results strongly suggest that INa and IA in vertebrate neurons are modulated by PKC; all PKC subtypes exert a similar effect on the A-channel while only subtypes II and III modulate the Na+ channel.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.     

Electrostatics of the intracellular vestibule of K+ channels

Previous calculations using continuum electrostatic calculations showed that a fully hydrated monovalent cation is electrostatically stabilized at the center of the cavity of the KcsA potassium channel. Further analysis demonstrated that this cavity stabilization was controlled by a balance between the unfavorable reaction field due to the finite size of the cavity and the favorable electrostatic field arising from the pore helices. In the present study, continuum electrostatic calculations are used to investigate how the stability of an ion in the intracellular vestibular cavity common to known potassium channels is affected as the inner channel gate opens and the cavity becomes larger and contiguous with the intracellular solution. The X-ray structure of the calcium-activated potassium channel MthK, which was crystallized in the open state, is used to construct models of the KcsA channel in the open state. It is found that, as the channel opens, the barrier at the helix bundle crossing decreases to approximately 0 kcal/mol, but that the ion in the cavity is also significantly destabilized. The results are compared and contrasted with additional calculations performed on the KvAP (voltage-activated) and KirBac1.1 (inward rectifier) channels, as well as models of the pore domain of Shaker in the open and closed state. In conclusion, electrostatic factors give rise to energetic constraints on ion permeation that have important functional consequences on the various K+ channels, and partly explain the presence or absence of charged residues near the inner vestibular entry.     

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.     

Voltage-gated Ca2+ channels in type III taste cells

In the mammalian taste bud, the heterogeneous cell population includes three morphologically distinct types of cells, type I to type III, which are also different in their electrophysiological features. Particularly, voltage-gated (VG) Ca²⁺ channels are functional solely in taste cells of the type III. These channels were studied here with external Ba²⁺ ions as current carriers. It was specifically shown that VG Ba²⁺ currents were almost completely blockable with nifedipine as well as with ionic blockers, such as Cd²⁺, Ni²⁺, and Co²⁺. Kinetic properties of VG Ba²⁺ currents in type III cells and their sensitivity to the blockers indicated that these currents were largely mediated by VG Ca²⁺ channels of the L-type. The expression of genes, which encode pore-forming α1-subunits of Ca²⁺ channels, was analyzed using methods of molecular biology. Among four genes encoding L-type Ca²⁺ channel α1-subunits (Ca _ν 1.1-Ca _ν 1.4), the expression of Ca _ν 1.2 was demonstrated in taste cells.