Increase In Open Probability Research Articles

Regions in the carboxy terminus of alpha-bENaC involved in gating and functional effects of actin.

Gating differences occur between the alpha-subunits of the bovine and rat clones of an amiloride-sensitive epithelial Na+ channel (ENaC). Deletion of the carboxy terminus of bovine alpha-ENaC (alpha-bENaC) at R567 converted the gating properties to that of rat alpha-ENaC (alpha-rENaC). The equivalent truncation in alpha-rENaC was without effect on the gating of the rat homologue. The addition of actin to ENaC channels composed of either alpha-rENaC or alpha-bENaC alone produced a twofold reduction in conductance and an increase in open probability. Neither alpha-rENaC (R613X) nor alpha-bENaC (R567X) was responsive to actin. Using a chimera consisting of alpha-rENaC1-615 and alpha-bENaC570-650, we examined several different carboxy-terminal truncation mutants plus and minus actin. When incorporated into planar bilayers, the gating pattern of this construct was identical to wild-type (wt) alpha-bENaC. Premature stop mutations proximal to E685X produced channels with gating patterns like alpha-rENaC. Actin had no effect on the E631X truncation, whereas more distal truncations all interacted with actin, as did wt alpha-bENaC. Key findings were confirmed using channels expressed in Xenopus oocytes and studied by cell-attached patch-clamp recording. Our results suggest that the site of actin regulation at the carboxy terminus of the chimera is located between residues 631 and 644.

Markovian Models of Low and High Activity Levels of Cardiac Ryanodine Receptors

The modal gating behavior of single sheep cardiac sarcoplasmic reticulum (SR) Ca 2+-release/ryanodine receptor (RyR) channels was assessed. We find that the gating of RyR channels spontaneously shifts between high (H) and low (L) levels of activity and inactive periods where no channel openings are detected (I). Moreover, we find that there is evidence for multiple gating modes within H activity, which we term H1 and H2 mode. Our results demonstrate that the underlying mechanisms regulating gating are similar in native and purified channels. Dwell-time distributions of L activity were best fitted by three open and five closed significant exponential components whereas dwell-time distributions of H1 activity were best fitted by two to three open and four closed significant exponential components. Increases in cytosolic [Ca 2+] cause an increase in open probability (Po) within L activity and an increase in the probability of occurrence of H activity. Open lifetime distributions within L activity were Ca 2+ independent whereas open lifetime distributions within H activity were Ca 2+ dependent. This study is the first attempt to estimate RyR single-channel kinetic parameters from sequences of idealized dwell-times and to develop kinetic models of RyR gating using the criterion of maximum likelihood. We propose distinct kinetic schemes for L, H1, and H2 activity that describe the major features of sheep cardiac RyR channel gating at these levels of activity.

Stimulation of cardiac L-type calcium channels by extracellular ATP.

The co-release of ATP with norepinephrine from sympathetic nerve terminals in the heart may augment adrenergic stimulation of cardiac Ca(2+) channel activity. To test for a possible direct effect of extracellular ATP on L-type Ca(2+) channels, single channels were reconstituted from porcine sarcolemma into planar lipid bilayers so that intracellular signaling pathways could be controlled. Extracellular ATP (2-100 microM) increased the open probability of the reconstituted channels, with a maximal increase of approximately 2.6-fold and an EC(50) of 3.9 microM. The increase in open probability was due to an increase in channel availability and a decrease in channel inactivation rate. Other nucleotides displayed a rank order of effectiveness of ATP > alpha,beta-methylene-ATP > 2-methylthio-ATP > UTP > adenosine 5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect. Several antagonists of P2 receptors had no impact on the ATP-dependent increase in open probability, indicating that receptor activation was not required. These results suggest that extracellular ATP and other nucleotides can stimulate the activity of cardiac L-type Ca(2+) channels via a direct interaction with the channels.

Do inactivation mechanisms rather than adaptation hold the key to understanding ryanodine receptor channel gating?

The use of flash photolysis of caged Ca2+ to rapidly elevate [Ca2+] at the cytosolic face of a cardiac ryanodine receptor (RyR) channel reconstituted into a bilayer gives rise to a rapid increase in open probability (Po; Gyorke and Fill 1993). This is followed by a decline in Po, which occurs much more slowly (τ = 1.3 s). After the decline in Po, the channels can be reactivated by a second Ca2+ stimulus. These results led to the suggestion that RyR channels can adapt to a maintained Ca2+ stimulus. The report was of great interest for two main reasons. First, the concept that Ca2+-induced Ca2+ release (CICR) was a process that was smoothly graded and dependent on the magnitude of the trigger rather than an all-or-nothing process was difficult to explain. Not long before the publication of the Gyorke and Fill report (1993), Stern 1992 had brought this issue to the forefront of discussions on excitation–contraction (EC) coupling in cardiac muscle. He elegantly described how global models of EC coupling needed to be discarded because they were inherently unstable and that “local control” models, where discrete units of RyR channels were activated individually, could be operating. Stern 1992 argued that diffusion of Ca2+ away from the Ca2+ activation sites on RyR, coupled with the process of “stochastic attrition,” led to the inactivation of the Ca2+ release units. The contraction of the whole- cell, therefore, resulted from the summation of the individual packets of Ca2+ released from the separate release units. Stern's local control model of cardiac EC coupling made sense of seemingly irreconcilable discrepancies and stimulated workers in the field to investigate, extend, or disprove his theory. It was at this time that Gyorke and Fill 1993 presented adaptation as a self-regulating property of RyR channels that could serve as a “molecular control mechanism for smoothly graded Ca2+-induced Ca2+-release in heart.” On the face of it, they had uncovered a mechanism that would close the RyR channels to allow reaccumulation of Ca2+ within sarcoplasmic reticulum (SR) stores, and that could explain why CICR was not an all-or-nothing positive feedback process. Gyorke and Fill's paper (1993) was closely followed by the first report of Ca2+ sparks by Cheng et al. 1993. The improved temporal and spatial resolution of in situ SR Ca2+-release events afforded by the use of the confocal microscope accelerated the integration of theoretical models of EC coupling with experimental data. Here was evidence that local control of individual Ca2+ release units could provide smoothly graded CICR. This was an exciting time for cardiac physiologists and biophysicists, and the idea that adaptation of RyR might refine existing models of EC coupling was appealing. The second reason for the interest in this work was the proposal that the phenomenon of adaptation was not merely the response of RyR to a rapid step change in [Ca2+], but that the Ca2+ spike that preceded the maintained increase in [Ca2+] itself affected channel gating (Lamb et al. 1994; Lamb and Laver 1998). Thus, Lamb and co-workers (Lamb et al. 1994; Lamb and Laver 1998) argued adaptation was the overall response of RyR channels to both the Ca2+ spike and the longer lasting increase in [Ca2+]. The idea that the Ca2+ spike influenced the occurrence of adaptation was fueled by subsequent reports that adaptation did not occur when rapid changes in cytosolic [Ca2+] were produced using methods other than flash photolysis of a caged Ca2+ compound (Schiefer et al. 1995; Sitsapesan et al. 1995; Laver and Curtis 1996). The issue was further confounded by the more recent report that Ca2+ spikes, in the absence of a maintained elevation in [Ca2+], could activate RyR channels (Zahradnikova et al. 1999), although previously Gyorke and Fill 1994 had claimed vigorously that Ca2+ spikes could not activate RyR channels. In fact, this was used as evidence indicating that the fast Ca2+ spike did not influence the effect of the more maintained change in [Ca2+] (Gyorke and Fill 1994). There are certain issues that need to be resolved before we can understand how RyR channels respond to rapid changes in [Ca2+], and before we can hope to understand how SR Ca2+ release is triggered and terminated. First, does the Ca2+ spike that precedes the maintained increase in [Ca2+] in flash photolysis experiments affect the subsequent gating behavior of RyR? Second, what is the response of the RyR channel to a maintained step increase in [Ca2+]? Finally, does the gating behavior of RyR channels and the evidence from triggered Ca2+ release in intact cells support the proposal that RyR channels adapt to a maintained increase in [Ca2+]?

Structural factors that determine the ability of adenosine and related compounds to activate the cardiac ryanodine receptor.

The effects of adenosine and adenine on the gating of native sheep cardiac ryanodine receptor (RyR) channels were investigated. By examining the mechanisms underlying channel activation and by using comparative molecular field analysis (CoMFA) we have investigated the structural features of adenine-based ligands involved in channel activation. In the presence of 10 microM cytosolic Ca(2+), adenosine and adenine both activate the channel but only to a level approximately 10 and 20% respectively of that of ATP indicating that both are partial agonists of low efficacy. Adenosine was able to antagonize the ATP-induced increase in open probability (Po) as expected for a partial agonist of low efficacy at the ATP sites on the cardiac RyR. GTP (100 microM - 10 mM) had no effect on channel gating indicating that the adenine ring structure is important for agonist activity at the ATP-sites on RyR. CoMFA revealed an extremely strong correlation between the structural features of the five ATP analogues and the ability to increase (Po). Our model indicates that the high efficacy of ATP results primarily from the large electrostatic field established by the ionized phosphate groups. Reducing the number of phosphate groups lowers the strength of this field, leading to ligands with lower efficacy. In addition, steric interactions between the alpha-phosphate and ribose moieties and the RyR are correlated with low Po.

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism.

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).

A new twist in the saga of charge movement in voltage-dependent ion channels.

Despite the many discrepancies in results and the different experimental methods used, both papers arrive at the same conclusion. S4 segments rotate when depolarized, and this rotation moves charged residues from an intracellular vestibule to an extracellular vestibule (Figure 2BFigure 2B). One reason this mechanism is so appealing is that the residues most important for charge transfer, the outermost four charged residues, lie along one face of an α helix. Note that in order for this mechanism to account for charge transfer, the direction of the electric field cannot be oriented parallel to the plane of the membrane, as implied for example by the shaded regions in Figure 1CFigure 1C, but more perpendicular to the membrane (Figure 2BFigure 2B). This reorientation of the electric field is a consequence of the topology of the aqueous vestibules surrounding the S4 segment.One prediction of these models is that the residues on the backside of this stripe of positive charge, i.e., the neutral residues between the charged residues in the primary sequence, should move in the opposite direction, from extracellular to intracellular during a depolarization. This does not occur. The residues between the second and third arginines in the primary sequence are in an intracellular compartment at hyperpolarized voltages (Figure 1CFigure 1C). This does not eliminate the possibility that S4 rotates. It means that the gating pore also undergoes conformational changes as the S4 segment moves. This fact is also apparent from accessibility studies, which suggest that the length of the gating pore changes with depolarization (Figure 1CFigure 1C).What message can we take home about S4 movement? As attractive as it is, the claim for a rotation as large as 180° is weak. Furthermore, even if a smaller rotation contributes to charge movement, it is likely to be only one of the movements of S4 and the protein surrounding it. Perhaps the most important conclusion supported by both studies is that the movements of the S4 segment are very small, as one might expect for a conformational change that has to be fast enough to underlie the gating currents.The raison d'etre of an S4 segment is not charge movement; it is the control of the activation gates at the bottom of the S6 segments. How are these movements coupled? One possibility is that movement of the S4 segment tugs or twists the linkers that connect it to S3 and S5 segments, and these segments transmit this movement to the S6 segment. Another possibility is that S4 and S6 are like entwined lovers. One cannot move without a compensatory rearrangement of the other. For example, S4 rotation may cause S6 rotation, a movement that underlies the gating of a bacterial potassium channel (Perozo et al. 1999xPerozo, E., Cortes, D.M., and Cuello, L.G. Science. 1999; 285: 73–78Crossref | PubMed | Scopus (461)See all ReferencesPerozo et al. 1999). The answers to these mysteries will surely unfold with the next episode of conformational studies of voltage-dependent gating.*E-mail: richard.horn@mail.tju.edu.

Mechanisms of increased Na(+) transport in ATII cells by cAMP: we agree to disagree and do more experiments.

Existing evidence supports the presence of active transport of Na(+) across the mammalian alveolar epithelium and its upregulation by agents that increase cytoplasmic cAMP levels. However, there is controversy regarding the mechanisms responsible for this upregulation. Herein we present the results of various patch-clamp studies indicating the presence of 25- to 27-pS, amiloride-sensitive, moderately selective Na(+) channels (Na(+)-to-K(+) permeability ratio = 7:1) located on the apical membranes of rat alveolar type II (ATII) cells maintained in primary culture. The addition of terbutaline to the bath solution increased the open probability of single channels present in cell-attached patches of ATII cells without affecting their conductance. A similar increase in open probability was seen after the addition of protein kinase A, ATP, and Mg(2+) to the cytoplasmic side of inside-out patches. Measurement of short-circuit currents across confluent monolayers of rat or rabbit ATII cells indicates that terbutaline and 8-(4-chlorophenylthio)-cAMP increase vectorial Na(+) transport and activate Cl(-) channels. Currently, there is a controversy as to whether the cAMP-induced increase in Na(+) transport is due solely to hyperpolarization of the cytoplasmic side of the ATII cell membrane due to Cl(-) influx or whether it results from simultaneous stimulation of both Cl(-) and Na(+) conductive pathways. Additional studies are needed to resolve this issue.

Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death.

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-D-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.

Effect of extracellular pH on GABA-activated current in rat recombinant receptors and thin hypothalamic slices.

We studied the effects of extracellular pH (pHo) on gamma-aminobutyric acid (GABA)-mediated Cl- current in rat hypothalamic neurons and recombinant type-A GABA (GABA(A)) receptors stably expressed in human embryonic kidney cells (HEK 293), using whole cell and outside-out patch-clamp recordings. In alpha3beta2gamma2s receptors, acidic pH decreased, whereas alkaline pH increased the response to GABA in a reversible and concentration-dependent manner. Acidification shifted the GABA concentration-response curve to the right, significantly increasing the EC50 for GABA without appreciably changing the slope or maximal current induced by GABA. We obtained similar effects of pH in alpha1beta2gamma2 receptors and in GABA-activated currents recorded from thin hypothalamic brain slices. In outside-out patches recorded from alpha3beta2gamma2 recombinant receptors, membrane patches were exposed to 5 microM GABA at control (7.3), acidic (6.4), or alkaline (8.4) pH. GABA activated main and subconductance states of 24 and 16 pS, respectively, in alpha3beta2gamma2 receptors. Alkaline pH(o) increased channel opening frequency and decreased the duration of the long closed state, resulting in an increase in open probability (from 0.0801 +/- 0.015 in pH 7.3 to 0.138 +/- 0.02 in pH 8.4). Exposure of the channels to acidic pH(o) had the opposite effects on open probability (decreased to 0.006 +/- 0.0001). Taken together, our results indicate that the function of GABA(A) receptors is modulated by extracellular pH. The proton effect is similar in recombinant and native receptors and is dependent on GABA concentration. In addition, the effect appears to be independent of the alpha-subunit isoform, and is due to the ability of H+ to alter the frequency of channel opening. Our findings indicate that GABAergic signaling in the CNS may be significantly altered during conditions that increase or decrease pH.

Evidence for novel caffeine and Ca2+ binding sites on the lobster skeletal ryanodine receptor

1. The effects of Ca2+, ATP and caffeine on the gating of lobster skeletal muscle ryanodine receptors (RyR) was investigated after reconstitution of the channels into planar phospholipid bilayers and by using [3H]-ryanodine binding studies. 2. The single channel studies reveal that the EC50 (60 microM) for activation of the lobster skeletal RyR by Ca2+ as the sole ligand is higher than for any other isoform of RyR studied. 3. Inactivation of the channel by Ca2+ (EC50 = 1 mM) occurs at concentrations slightly higher than those required to inactivate mammalian skeletal RyR (RyR1) but lower than those required to inactivate mammalian cardiac RyR (RyR2). 4. Lifetime analysis demonstrates that cytosolic Ca2+, as the sole activating ligand, cannot fully open the lobster skeletal RyR (maximum Po approximately 0.2). The mechanism for the increase in open probability (Po) is an increase in both the frequency and the duration of the open events. 5. ATP is a very effective activator of the lobster RyR and can almost fully open the channel in the presence of activating cytosolic [Ca2+]. In the presence of 700 microM Ca2+, 1 mM ATP increased Po to approximately 0.8. 6. Caffeine, often used as a tool to identify the presence of RyR channels, is relatively ineffective and cannot increase Po above the level that can be attained with Ca2+ alone. 7. The results reveal that caffeine increases Po by a different mechanism to that of cytosolic Ca2+ demonstrating that the mechanism for channel activation by caffeine is not 'sensitization' to cytosolic Ca2+. 8. By studying the mechanisms involved in the activation of the lobster RyR we have demonstrated that the channel responds in a unique manner to Ca2+ and to caffeine. The results strongly indicate that these ligand binding sites on the channel are different to those on mammalian isoforms of RyR.

Peptide inhibition of ENaC.

Liddle's disease is an autosomal dominant form of human hypertension resulting from a basal activation of amiloride-sensitive Na+ channels (ENaC). This channel activation is produced by mutations in the beta- and/or gamma-carboxy-terminal cytoplasmic tails, in many cases causing a truncation of the last 45-76 amino acids. In this study, we tested two hypotheses; first, beta- and gamma-ENaC C-terminal truncation mutants (beta DeltaC and gamma DeltaC), in combination with the wild-type alpha-ENaC subunit, reproduce the Liddle's phenotype at the single channel level, i.e., an increase in open probability (Po), and second, these C-terminal regions of beta- and gamma-ENaC act as intrinsic blockers of this channel. Our results indicate that alpha beta DeltaC gamma DeltaC-rENaC, incorporated into planar lipid bilayers, has a significantly higher single channel Po compared to the wild-type channel (0.85 vs 0.60, respectively), and that 30-mer synthetic peptides corresponding to the C-terminal region of either beta- or gamma-ENaC block the basal-activated channel in a concentration-dependent fashion. Moreover, there was a synergy between the peptides for channel inhibition when added together. We conclude that the increase in macroscopic Na+ reabsorption that occurs in Liddle's disease is at least in part due to an increase in single channel Po and that the cytoplasmic tails of the beta- and gamma-ENaC subunits are important in the modulation of ENaC activity.

Carboxylmethylation of the β Subunit of xENaC Regulates Channel Activity

The action of aldosterone to increase apical membrane permeability in responsive epithelia is thought to be due to activation of sodium channels. Aldosterone stimulates methylation of a 95-kDa protein in apical membrane of A6 cells, and we have previously shown that methylation of a 95-kDa protein in the immunopurified Na+ channel complex increases open probability of these channels in planar lipid bilayers. We report here that aldosterone stimulates carboxylmethylation of the beta subunit of xENaC in A6 cells. In vitro translated beta subunit, but not alpha or gamma, serves as a substrate for carboxylmethylation. Carboxylmethylation of ENaC reconstituted in planar lipid bilayers leads to an increase in open probability only when beta subunit is present. When the channel complex is immunoprecipitated from A6 cells and analyzed by Western blot with antibodies to the three subunits of xENaC, all three subunits are recognized as constituents of the complex. The results suggest that Na+ channel activity in A6 cells is regulated, in part, by carboxylmethylation of the beta subunit of xENaC.

Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messenger-induced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the "afterdischarge. " In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10. 7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5'-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium/phospholipid-dependent protein kinase C (PKC) inhibitor peptide(19-36). Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.

Small-conductance Ca(2+)-dependent K+ channels activated by ATP in murine colonic smooth muscle.

The patch-clamp technique was used to determine the ionic conductances activated by ATP in murine colonic smooth muscle cells. Extracellular ATP, UTP, and 2-methylthioadenosine 5'-triphosphate (2-MeS-ATP) increased outward currents in cells with amphotericin B-perforated patches. ATP (0.5-1 mM) did not affect whole cell currents of cells dialyzed with solutions containing ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Apamin (3 x 10(-7) M) reduced the outward current activated by ATP by 32 +/- 5%. Single channel recordings from cell-attached patches showed that ATP, UTP, and 2-MeS-ATP increased the open probability of small-conductance, Ca(2+)-dependent K+ channels with a slope conductance of 5.3 +/- 0.02 pS. Caffeine (500 microM) enhanced the open probability of the small-conductance K+ channels, and ATP had no effect after caffeine. Pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS, 10(-4) M), a nonselective P2 receptor antagonist, prevented the increase in open probability caused by ATP and 2-MeS-ATP. PPADS had no effect on the response to caffeine. ATP-induced hyperpolarization in the murine colon may be mediated by P2y-induced release of Ca2+ from intracellular stores and activation of the 5.3-pS Ca(2+)-activated K+ channels.

Mechanisms of modulation by internal protons of cyclic nucleotide-gated channels cloned from sensory receptor cells.

We have examined the modulation by internal protons of cyclic nucleotide-gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside-out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit 1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single-channel current, consistent with protonation of a single acidic site with a pK1 of 4.5-4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single-channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel.

A large conductance, Ca 2+-activated K + channel in a human lung epithelial cell line (A549)

A large conductance, Ca 2+-activated K + channel in a human lung epithelial cell line (A549) was identified using the single channel patch clamp technique. Channel conductance was 242±33 pS ( n=67) in symmetrical KCl (140 mM). The channel was activated by membrane depolarization and increased cytosolic Ca 2+. High selectivity was observed for K + over Rb +(0.49)>Cs +(0.14)>Na +(0.09). Open probability was significantly decreased by Ba 2+ (5 mM) and quinidine (5 mM) to either surface, but TEA (5 mM) was only effective when added to the external surface. All effects were reversible. Increasing cytosolic Ca 2+ concentration from 10 −7 to 10 −6 M caused an increase in open probability from near zero to fully activated. ATP decreased open probability at ∼2 mM, but the effect was variable. The channel was almost always observed together with a smaller conductance channel, although they could both be seen individually. We conclude that A549 cells contain large conductance Ca 2+-activated K + channels which could explain a major fraction of the K + conductance in human alveolar epithelial membranes.

Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers

Protein kinase A (PKA)- and G protein-mediated regulation of immunopurified adult rabbit alveolar epithelial type II (ATII) cell proteins that exhibit amiloride-sensitive Na+ channel activity was studied in planar lipid bilayers and freshly isolated ATII cells. Addition of the catalytic subunit of PKA + ATP increased single channel open probability from 0.42 +/- 0.05 to 0.82 +/- 0.07 in a voltage-independent manner, without affecting unitary conductance. This increase in open probability of the channels was mainly due to a decrease in the time spent by the channel in its closed state. The apparent inhibition constant for amiloride increased from 8.0 +/- 1.8 microM under control conditions to 15 +/- 3 microM after PKA-induced phosphorylation; that for ethylisopropylamiloride increased from 1.0 +/- 0.4 to 2.0 +/- 0.5 microM. Neither pertussis toxin (PTX) nor guanosine 5'-O-(3-thiotriphosphate) affected ATII Na+ channel activity in bilayers. Moreover, PTX failed to affect amiloride-inhibitable 22Na+ uptake in freshly isolated ATII cells. In vitro, ADP ribosylation induced by PTX revealed the presence of a specifically ribosylated band at 40-45 kDa in the total solubilized ATII cell protein fraction, but not in the immunopurified fraction. Moreover, the immunopurified channel was downregulated in response to guanosine 5'-O-(3-thiotriphosphate)-mediated activation of the exogenous G alpha(i-2), but not G(oA), G alpha(i-1), or G alpha(i-3), protein added to the channel. This effect occurred only in the presence of actin. These results suggest that amiloride-sensitive Na+ channels in adult alveolar epithelia regulated by PKA-mediated phosphorylation also retain the ability to be regulated by G alpha([i-2), but not G alpha([i-1) or G alpha(i-3), protein.

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A.

We identified voltage-activated K+ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca2+-activated K+ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 +/- 1.7 pS channels (KDR1), 90.6 +/- 5.4 pS channels (KDR2) and 149 +/- 4 pS intermediate-conductance Ca2+-activated K+ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from KDR1 channels inactivated with a time constant of 1.7 +/- 0.1 s at +60 mV test potential, while KDR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca2+] (10(-6 )M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). KDR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1-10 mM) but not to Ca2+. KDR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of KDR1 channels 3.4-fold and of KDR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (-60 mV) only openings of KDR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K+ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells.

Regulation of Epithelial Sodium Channels by Short Actin Filaments

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.