Regulation of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are found in several cell types, and are best studied in photoreceptors and olfactory sensory neurons. There, CNG channels are gated by the second messengers of the visual and olfactory signalling cascades, cGMP and cAMP respectively, and operate as transduction channels generating the stimulus-induced receptor potentials. In visual and olfactory sensory cells CNG channels conduct cationic currents. Calcium can contribute a large fraction of this current, and calcium influx serves a modulatory role in CNG-channel mediated signal transduction. There have been recent developments in our understanding of how the regulation of CNG channels contributes to the physiological properties of photoreceptors and olfactory sensory cells, and in particular on the role of calcium-mediated feedback.     

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.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Irreversible block of cardiac mutant Na+ channels by batrachotoxin

Batrachotoxin (BTX) not only keeps the voltage-gated Na(+) channel open persistently but also reduces its single-channel conductance. Although a BTX receptor has been delimited within the inner cavity of Na(+) channels, how Na(+) ions flow through the BTX-bound permeation pathway remains unclear. In this report we tested a hypothesis that Na(+) ions traverse a narrow gap between bound BTX and residue N927 at D2S6 of cardiac hNa(v)1.5 Na(+) channels. We found that BTX at 5 microM indeed elicited a strong block of hNa(v)1.5-N927K currents (approximately 70%) after 1000 repetitive pulses (+50 mV/20 ms at 2 Hz) without any effects on Na(+) channel gating. Once occurred, this unique use-dependent block of hNa(v)1.5-N927K Na(+) channels recovered little at holding potential (-140 mV), demonstrating that BTX block is irreversible under our experimental conditions. Such an irreversible effect likewise developed in fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels albeit with a faster on-rate; approximately 90% of peak Na(+) currents were abolished by BTX after 200 repetitive pulses (+50 mV/20 ms). This use-dependent block of fast inactivation-deficient hNa(v)1.5-N927K Na(+) channels by BTX was duration dependent. The longer the pulse duration the larger the block developed. Among N927K/W/R/H/D/S/Q/G/E substitutions in fast inactivation-deficient hNa(v)1.5 Na(+) channels, only N927K/R Na(+) currents were highly sensitive to BTX block. We conclude that (a) BTX binds within the inner cavity and partly occludes the permeation pathway and (b) residue hNa(v)1.5-N927 is critical for ion permeation between bound BTX and D2S6, probably because the side-chain of N927 helps coordinate permeating Na(+) ions.     

A multiply charged tetracaine derivative blocks cyclic nucleotide-gated channels at subnanomolar concentrations

Cyclic nucleotide-gated (CNG) ion channels are central participants in sensory transduction, generating the electrical response to light in retinal photoreceptors and to odorants in olfactory receptors. They are expressed in many other tissues where their specific roles in signaling remain unclear. As is true for many other ion channels, there is a paucity of specific blockers needed to dissect the contributions of these channels to cell signaling. CNG channels are members of the superfamily of voltage-gated ion channels, and the local anesthetic tetracaine is known to block CNG channels in a manner that resembles the block of voltage-gated Na(+) channels. The amine in local anesthetics interacts with the charged selectivity filter of Na(+) channels, while the aromatic ring gets stuck in the inner cavity and has hydrophobic interactions with the residues lining that region. Here we have synthesized a derivative of tetracaine, 3-[(aminopropyl)amino]-N,N-dimethyl-N-(2-[[4-(butylamino)benzoyl]oxy]ethyl)propan-1-aminium acetate (APPA-tetracaine), that contains three positively charged amines at physiological pH instead of one. This compound blocked several different CNG channels in the picomolar to nanomolar concentration range at positive membrane potentials, making it several orders of magnitude more potent than tetracaine. In contrast, significant block of Na(+) channels by APPA-tetracaine required concentrations of hundreds of nanomolar. The results suggest that the highly charged moiety of APPA-tetracaine interacts strongly with the negative charge cluster in the selectivity filter of CNG channels. We propose that a variety of potent and specific ion channel blockers could be generated by expanding on traditional blocker structures to target the selectivity filters of other channels.     

Intracellular Ca2+ regulates the sensitivity of cyclic nucleotide-gated channels in olfactory receptor neurons.

In olfactory receptor neurons, odorants stimulate production of cAMP, which directly activates cyclic nucleotide-gated (CNG) channels. Olfactory adaptation is thought to result from a rise in intracellular Ca2+. To determine whether inhibition of CNG channels plays a role in adaptation, we have investigated the action of Ca2+ on these channels in inside-out "macro" patches from the dendrite and cilia of catfish olfactory neurons. Internal Ca2+, with a K1/2 of 3 microM, profoundly inhibits CNG channels by shifting the dose-response relationship to higher cAMP levels without altering the maximal response. The inhibition does not appear to result from a direct interaction of Ca2+ with the CNG channel. Thus, the inhibition washes out after excision of the inside-out patch, and Ca2+ does not inhibit the cloned catfish CNG channel expressed in Xenopus oocytes. Hence we propose that a regulatory Ca(2+)-binding protein, distinct from the CNG channel, controls the gain of signal transduction and contributes to olfactory adaptation by decreasing the sensitivity of the CNG channel to cAMP.     

Sodium fluxes through nonselective cation channels in the plasma membrane of protoplasts from Arabidopsis roots

The aim of the present work was to characterize Na(+) currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca(2+) by digesting tissue in elevated Ca(2+). Experiments in whole-cell and outside-out modes were carried out. We found that Na(+) currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium(+) and verapamil, had no time-dependent activation (permanently opened or completely activated within 1-2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K(+) (1.49) > NH(4)(+) (1.24) > Rb(+) (1.15) approximately equal to Cs(+) (1.10) approximately equal to Na(+) (1.00) > Li(+) (0.73) > tetraethylammonium(+) (0.47). Arabidopsis root NSCCs were blocked by H(+) (pK approximately equal to 6.0), Ca(2+) (K(1/2) approximately equal to 0.1 mM), Ba(2+), Zn(2+), La(3+), Gd(3+), quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca(2+)-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na(+) influx to the cell and probably having other important roles in physiological processes of plants.     

Mechanisms of cation permeation in cardiac sodium channel: description by dynamic pore model.

The selective permeability to monovalent metal cations, as well as the relationship between cation permeation and gating kinetics, was investigated for native tetrodotoxin-insensitive Na-channels in guinea pig ventricular myocytes using the whole-cell patch clamp technique. By the measurement of inward unidirectional currents and biionic reversal potentials, we demonstrate that the cardiac Na-channel is substantially permeable to all of the group Ia and IIIa cations tested, with the selectivity sequence Na(+) >/= Li(+) > Tl(+) > K(+) > Rb(+) > Cs(+). Current kinetics was little affected by the permeant cation species and concentrations tested (相似文献   

Ion Permeation and Selectivity of Wild-Type Recombinant Rat CNG (rOCNC1) Channels Expressed in HEK293 Cells

The permeation properties of adenosine 3′, 5′-cyclic monophosphate (cAMP)-activated recombinant rat olfactory cyclic nucleotide-gated channels (rOCNC1) in human embryonic kidney (HEK 293) cells were investigated using inside-out excised membrane patches. The relative permeability of these rOCNC1 channels to monovalent alkali cations and organic cations was determined from measurements of the changes in reversal potential upon replacing sodium in the bathing solution with different test cations. The permeability ratio of Cl⁻ relative to Na⁺ (P _Cl /P _Na ) was about 0.14, confirming that these channels are mainly permeable to cations. The sequence of relative permeabilities of monovalent alkali metal ions in these channels was P _Na ≥P _K > P _Li > P _Cs ≥P _Rb , which closely corresponds to a high-strength field sequence as previously determined for native rat olfactory receptor neurons (ORNs). The permeability sequence for organic cations relative to sodium was P _NH3OH > P _NH4 > P _Na > P _Tris > P _Choline > P _TEA , again in good agreement with previous permeability ratios obtained in native rat ORNs. Single-channel conductance sequences agreed surprisingly well with permeability sequences. These conductance measurements also indicated that, even in asymmetric bi-ionic cation solutions, the conductance was somewhat independent of current direction and dependent on the composition of both solutions. These results indicate that the permeability properties of rOCNC1 channels are similar to those of native rat CNG channels, and provide a suitable reference point for exploring the molecular basis of ion selectivity in recombinant rOCNC1 channels using site-directed mutagenesis. Received: 3 July 2000/Revised: 29 August 2000     

Concentration Dependence of Sodium Permeation and Sodium Ion Interactions in the Cyclic AMP-gated Channels of Mammalian Olfactory Receptor Neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 μm) of adenosine 3′, 5′-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mm. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mm and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na⁺ equilibrium potential indicating that P _Cl/P _Na≈ 0. However, at low external NaCl concentrations (≤100 mm) there was some significant chloride permeability. Our results further indicated that Na⁺ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K _ms in the range of 100–150 mm and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels. Received: 7 November 1996/Revised: 24 March 1997     

Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pacemaker channels produce an instantaneous current.

Spontaneous rhythmic activity in mammalian heart and brain depends on pacemaker currents (I(h)), which are produced by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here, we report that the mouse HCN2 pacemaker channel isoform also produced a large instantaneous current (I(inst(HCN2))) in addition to the well characterized, slowly activating I(h). I(inst(HCN2)) was specific to expression of HCN2 on the plasma membrane and its amplitude was correlated with that of I(h). The two currents had similar reversal potentials, and both were modulated by changes in intracellular Cl(-) and cAMP. A mutation in the S4 domain of HCN2 (S306Q) decreased I(h) but did not alter I(inst(HCN2)), and instantaneous currents in cells expressing either wild type HCN2 or mutant S306Q channels were insensitive to block by Cs(+). Co-expression of HCN2 with the accessory subunit, MiRP1, decreased I(h) and increased I(inst(HCN2)), suggesting a mechanism for modulation of both currents in vivo. These data suggest that expression of HCN channels may be accompanied by a background conductance in native tissues and are consistent with at least two open states of HCN channels: I(inst(HCN2)) is produced by a Cs(+)-open state; hyperpolarization produces an additional Cs(+)-sensitive open state, which results in I(h).     

Ca2+ permeation in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

Modulation of the two-pore domain acid-sensitive K+ channel TASK-2 (KCNK5) by changes in cell volume

The molecular identity of K(+) channels involved in Ehrlich cell volume regulation is unknown. A background K(+) conductance is activated by cell swelling and is also modulated by extracellular pH. These characteristics are most similar to those of newly emerging TASK (TWIK-related acid-sensitive K(+) channels)-type of two pore-domain K(+) channels. mTASK-2, but not TASK-1 or -3, is present in Ehrlich cells and mouse kidney tissue from where the full coding sequences were obtained. Heterologous expression of mTASK-2 cDNA in HEK-293 cells generated K(+) currents in the absence intracellular Ca(2+). Exposure to hypotonicity enhanced mTASK-2 currents and osmotic cell shrinkage led to inhibition. This occurred without altering voltage dependence and with only slight decrease in pK(a) in hypotonicity but no change in hypertonicity. Replacement with other cations yields a permselectivity sequence for mTASK-2 of K(+) > Rb(+) Cs(+) > NH(4)(+) > Na(+) congruent with Li(+), similar to that for the native conductance (I(K, vol)). Clofilium, a quaternary ammonium blocker of I(K, vol), blocked the mTASK-2-mediated K(+) current with an IC(50) of 25 microm. The presence of mTASK-2 in Ehrlich cells, its functional similarities with I(K, vol), and its modulation by changes in cell volume suggest that this two-pore domain K(+) channel participates in the regulatory volume decrease phenomenon.     

cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.     

Channels involved in transient currents unmasked by removal of extracellular calcium in cardiac cells

In cardiac cells that lack macroscopic transient outward K(+) currents (I(to)), the removal of extracellular Ca(2+) can unmask "I(to)-like" currents. With the use of pig ventricular myocytes and the whole cell patch-clamp technique, we examined the possibility that cation efflux via L-type Ca(2+) channels underlies these currents. Removal of extracellular Ca(2+) and extracellular Mg(2+) induced time-independent currents at all potentials and time-dependent currents at potentials greater than -50 mV. Either K(+) or Cs(+) could carry the time-dependent currents, with reversal potential of +8 mV with internal K(+) and +34 mV with Cs(+). Activation and inactivation were voltage dependent [Boltzmann distributions with potential of half-maximal value (V(1/2)) = -24 mV and slope = -9 mV for activation; V(1/2) = -58 mV and slope = 13 mV for inactivation]. The time-dependent currents were resistant to 4-aminopyridine and to DIDS but blocked by nifedipine at high concentrations (IC(50) = 2 microM) as well as by verapamil and diltiazem. They could be increased by BAY K-8644 or by isoproterenol. We conclude that the I(to)-like currents are due to monovalent cation flow through L-type Ca(2+) channels, which in pig myocytes show low sensitivity to nifedipine.     

AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers

Transepithelial transport of Na(+) across the lung epithelium via amiloride-sensitive Na(+) channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na(+) conductance (G(Na+)) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G(Na+): a 5-pS highly Na(+) selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to -38 mV and 0 mV to -35 mV. Amiloride at 1 microM inhibited HSC activity and 56% of short-circuit current (I(sc)), whereas 10 microM amiloride partially reduced NSC activity and inhibited a further 30% of I(sc). Neither conductance was associated with CNG channels as there was no effect of 10 microM pimoside on I(sc), HSC, or NSC activity, and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na(+)-permeable channels by a mechanism that likely reduces channel open probability.