Enhanced Channel Activity Research Articles

Contribution of the Kir3.1 Subunit to the Muscarinic-gated Atrial Potassium Channel IKACh

The muscarinic-gated atrial potassium (I(KACh)) channel contributes to the heart rate decrease triggered by the parasympathetic nervous system. I(KACh) is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits, although Kir3.4 homomultimers have also been proposed to contribute to this conductance. While Kir3.4 homomultimers evince many properties of I(KACh), the contribution of Kir3.1 to I(KACh) is less well understood. Here, we explored the significance of Kir3.1 using knock-out mice. Kir3.1 knock-out mice were viable and appeared normal. The loss of Kir3.1 did not affect the level of atrial Kir3.4 protein but was correlated with a loss of carbachol-induced current in atrial myocytes. Low level channel activity resembling recombinant Kir3.4 homomultimers was observed in 40% of the cell-attached patches from Kir3.1 knock-out myocytes. Channel activity typically ran down quickly, however, and was not recovered in the inside-out configuration despite the addition of GTP and ATP to the bath. Both Kir3.1 knock-out and Kir3.4 knock-out mice exhibited mild resting tachycardias and blunted responses to pharmacological manipulation intended to activate I(KACh). We conclude that Kir3.1 confers properties to I(KACh) that enhance channel activity and that Kir3.4 homomultimers do not contribute significantly to the muscarinic-gated potassium current.

H2O2 activates ryanodine receptor but has little effect on recovery of releasable Ca2+ content after fatigue.

We studied whether hydrogen peroxide (H(2)O(2)) at </=10 microM activates the ryanodine receptor and decreases releasable Ca(2+) content in the sarcoplasmic reticulum after fatigue. Exposure of rabbit or frog skeletal muscle ryanodine receptors to 10 microM H(2)O(2) enhanced channel activity in lipid bilayers when the redox potential was defined at cis = -220 mV and trans = -180 mV. Channel activation by 10 microM H(2)O(2) was also observed when cis potential was set at -220 mV without defining trans potential, but the effect was less. Reduction of trans redox potential from -180 to -220 mV did not alter channel activity. H(2)O(2) at 500 microM failed to activate the channel when the redox potential was not controlled. Stimulation of the frog muscle fiber for 2 min (50 Hz, a duty cycle of 200 ms/s) decreased tetanus tension by approximately 50%. After 1 min, tetanus recovered rapidly to approximately 70% of control and thereafter slowly approached the control level. Amplitudes of caffeine- and 4-chloro-m-cresol-induced contractures were decreased after a 60-min rest. The decrease is not enhanced by exposure to 10 microM H(2)O(2). These results suggest that H(2)O(2) markedly activates the ryanodine receptor under the redox control in vitro, but externally applied H(2)O(2) may not play an important role in the postfatigue recovery process.

Alterations in Insulin-Like Growth Factor-I Signaling in Cardiomyocytes From Chronic Alcohol-Exposed Rats

Background Insulin-like growth factor-I (IGF-I) is a required cytokine for the development and maintenance of the cardiovascular system, and it may play a role in certain pathophysiological conditions. Methods Adult male rats were fed a liquid diet that contained 36% alcohol for 4 to 8 months, and their littermates served as isocaloric pair-fed controls, so we could examine IGF-I signaling in rat cardiomyocyte preparations. Results Recently, our laboratory reported that IGF-I activates protein kinase-C (PKC)-α and that PKC-α activity is required for IGF-I-dependent activation of Erk1/Erk2 and IGF-I-dependent protein synthesis. But in chronic alcohol-exposed rats, there is loss of PKC-α activation by IGF-I, represented as a reduction in PKC-α translocation to the membrane. In reverse transcription-polymerase chain reaction experiments, both the alcoholic and control animals expressed the IGF-I receptor (IGF-1R, α subunit) and IGF-I mRNAs in approximately equal amounts. However, in the alcoholic cardiomyocyte protein preparations, there was a higher basal level of IGF-1R autophosphorylation of its internal tyrosine kinase domain, and IGF-I-activated autophosphorylation was reduced in the alcoholic protein preparations. Previously, we have demonstrated that acute IGF-I exposure enhances the rate of Mn2+ influx through activated nitrendipine-sensitive cardiac Ca2+ channels, and that this enhancement of channel activity is PKC-dependent. Here, we report that in alcohol-exposed myocytes, IGF-I-induced augmentation of the cardiac Ca2+ channel activity was absent. IGF-I increased the rate of protein synthesis in the control animals by 56% as determined in protein synthesis experiments that measured the rate of 14C-L-phenylalanine incorporation over time, and this effect was blocked by preincubation with Gö6976, a specific inhibitor of PKC-α. However, in the alcohol-exposed cardiomyocytes, IGF-I did not increase the rate of protein synthesis. Conclusion These results suggest that IGF-I-dependent PKC-α activation and IGF-I-dependent protein synthesis are altered in the hearts of chronic alcohol-exposed rats. This may be the result of the IGF-1R being in a chronically activated state, and it may alter the normal function of PKC-α.

Alterations in Insulin‐Like Growth Factor‐I Signaling in Cardiomyocytes From Chronic Alcohol‐Exposed Rats

Background Insulin‐like growth factor‐I (IGF‐I) is a required cytokine for the development and maintenance of the cardiovascular system, and it may play a role in certain pathophysiological conditions.Methods Adult male rats were fed a liquid diet that contained 36% alcohol for 4 to 8 months, and their littermates served as isocaloric pair‐fed controls, so we could examine IGF‐I signaling in rat cardiomyocyte preparations.Results Recently, our laboratory reported that IGF‐I activates protein kinase‐C (PKC)‐α and that PKC‐α activity is required for IGF‐I‐dependent activation of Erk1/Erk2 and IGF‐I‐dependent protein synthesis. But in chronic alcohol‐exposed rats, there is loss of PKC‐α activation by IGF‐I, represented as a reduction in PKC‐α translocation to the membrane. In reverse transcription‐polymerase chain reaction experiments, both the alcoholic and control animals expressed the IGF‐I receptor (IGF‐1R, α subunit) and IGF‐I mRNAs in approximately equal amounts. However, in the alcoholic cardiomyocyte protein preparations, there was a higher basal level of IGF‐1R autophosphorylation of its internal tyrosine kinase domain, and IGF‐I‐activated autophosphorylation was reduced in the alcoholic protein preparations. Previously, we have demonstrated that acute IGF‐I exposure enhances the rate of Mn2+ influx through activated nitrendipine‐sensitive cardiac Ca2+ channels, and that this enhancement of channel activity is PKC‐dependent. Here, we report that in alcohol‐exposed myocytes, IGF‐I‐induced augmentation of the cardiac Ca2+ channel activity was absent. IGF‐I increased the rate of protein synthesis in the control animals by 56% as determined in protein synthesis experiments that measured the rate of 14C‐L‐phenylalanine incorporation over time, and this effect was blocked by preincubation with Gö6976, a specific inhibitor of PKC‐α. However, in the alcohol‐exposed cardiomyocytes, IGF‐I did not increase the rate of protein synthesis.Conclusion These results suggest that IGF‐I‐dependent PKC‐α activation and IGF‐I‐dependent protein synthesis are altered in the hearts of chronic alcohol‐exposed rats. This may be the result of the IGF‐1R being in a chronically activated state, and it may alter the normal function of PKC‐α.

Mrp2 modulates the activity of chloride channels in isolated hepatocytes

Adenosine triphosphate binding cassette family transport proteins are important organic ion transporters in hepatocytes but these molecules may also exhibit other functions. In the present study we have measured the effects of substrates of the canalicular organic ion transporter multidrug resistance associated protein 2 (Mrp2) on chloride channel activation and cell volume regulation. We found that substrates such as leukotriene D 4, 17-β-estradiol glucuronide, and the leukotriene inhibitor MK-571 accelerated the activation of chloride channels by cell swelling and activated chloride channels in cytokine-pretreated hepatocytes. Two conjugated estrogens that are not Mrp2 substrates did not produce this effect. Hepatocytes derived from a strain of transport-deficient rats (TR −), which lack Mrp2 expression, showed none of these substrate effects. Coincident with their ability to activate channels, the Mrp2 substrates increased the rate of volume regulatory decrease by approximately 50% ( P < .01), confirming that enhanced channel activation under this condition stimulated volume regulation. In TR-hepatocytes the Mrp2 substrate had no effect on volume regulation. In conclusion, Mrp2 plays a role in regulation of chloride channel function by reducing the lag time necessary for channel activation and consequently accelerating the process of cell volume regulation. Substrates of Mrp2 affect the ability of the protein to interact with chloride channels. These findings represent an alternative function of Mrp2 in hepatocytes. (H EPATOLOGY 2002;36:65-71.)

Gβ Residues That Do Not Interact with Gα Underlie Agonist-independent Activity of K+ Channels

Gbetagamma subunits interact directly and activate G protein-gated Inwardly Rectifying K(+) (GIRK) channels. Little is known about the identity of functionally important interactions between Gbetagamma and GIRK channels. We tested the effects of all mammalian Gbeta subunits on channel activity and showed that whereas Gbeta1-4 subunits activate heteromeric GIRK channels independently of receptor activation, Gbeta5 does not. Gbeta1 and Gbeta5 both bind the N and C termini of the GIRK1 and GIRK4 channel subunits. Chimeric analysis between the Gbeta1 and Gbeta5 proteins revealed a 90-amino acid stretch that spans blades two and three of the seven-propeller structure and is required for channel activation. Within this region, eight non-conserved amino acids were critical for the activity of Gbeta1, as mutation of each residue to its counterpart in Gbeta5 significantly reduced the ability of Gbeta1 to stimulate channel activity. In particular, mutation of residues Ser-67 and Thr-128 to the corresponding Gbeta5 residues completely abolished Gbeta1 stimulation of GIRK channel activity. Mapping these functionally important residues on the three-dimensional structure of Gbeta1 shows that Ser-67, Ser-98, and Thr-128 are the only surface accessible residues. Galpha(i)1 interacts with Ser-98 but not with Ser-67 and Thr-128 in the heterotrimeric Galphabetagamma structure. Further characterization of the three mutant proteins showed that they fold properly and interact with Ggamma2. Of the three identified functionally important residues, the Ser-67 and Thr-128 Gbeta mutants significantly inhibited basal currents of a channel point mutant that displays Gbetagamma-mediated basal but not agonist-induced currents. Our findings indicate that the presence of Gbeta residues that do not interact with Galpha are involved in Gbetagamma interactions in the absence of agonist stimulation.

Regulation of epithelial sodium channel activity through a region of the carboxyl terminus of the alpha -subunit. Evidence for intracellular kinase-mediated reactions.

The epithelial sodium channel (ENaC) is a heteromultimer composed of three subunits, each having two membrane-spanning domains with intracellular amino and carboxyl termini. Several hormones and proteins regulate channel activity, but the molecular nature of this regulation is unknown. We conducted experiments to determine a possible new site within the carboxyl terminus of the alpha-subunit involved in enhanced channel activity through endogenous kinases. When an alpha-subunit that was truncated to remove a PY motif was expressed in Xenopus oocytes with wild type human beta- and gamma-ENaC subunits, channel activity was greatly enhanced. The removal of the entire intracellular carboxyl terminus of the alpha-subunit eliminated this enhanced basal activity. Using several point mutations, we localized this site to two amino acid residues (Pro(595)-Gly(596)) near the second membrane-spanning domain. The nonspecific kinase inhibitor staurosporine inhibits basal channel activity of wild type ENaC but was ineffective in inhibiting channels mutated at this site. The major effect of these mutations was not on channel kinetics but was largely, if not entirely, on the number of active channels on the cell surface. This region is potentially important in effecting kinase-mediated increases in ENaC activity.

Trifluoroacetic Acid Activates ATP-Sensitive K+ Channels in Rabbit Ventricular Myocytes

Recent in vivo experimental evidence suggests that isoflurane-induced cardioprotection may involve KATP channel activation during myocardial ischemia. The actual effect of isoflurane on cardioprotective ion conductance, however, such as that mediated by the opening of KATP channels, has been the subject of some controversy in the past. The investigation reported here used a patch-clamp technique to test the hypothesis that a metabolite of isoflurane, trifluoroacetic acid (TFA), contributes to isoflurane-induced cardioprotection via KATP channel activation. TFA enhanced channel activity in a concentration-dependent fashion, exhibiting half-maximal activation at 0.03 mM. TFA increased the number of openings of the channel, but did not affect the single channel conductance of KATP channels. Analysis of open and closed time distributions showed that TFA increased the burst duration and decreased the interburst interval without eliciting changes of less than 5 ms in open and closed time distributions. TFA diminished the ATP sensitivity of KATP channels in a concentration–response relationship for ATP. These results imply that TFA could mediate isoflurane-induced cardioprotection via KATP channel activation during myocardial ischemia and reperfusion.

Voltage-Induced Activation of Mechanosensitive Cation Channels of Leech Neurons

The voltage dependence of stretch-activated cation channels in leech central neurons was studied in cell-free configurations of the patch-clamp technique. We established that stretch-activated channels excised from identified cell bodies of desheathed ganglia, as well as from neurons in culture, were slowly and reversibly activated by depolarizing membrane potentials. Negative pressure stimuli, applied to the patch pipette during a slow periodical modulation of membrane potential, enhanced channel activity, whereas positive pressures depressed it. Voltage-induced channel activation was observed, with soft glass pipettes, both in inside-out and outside-out membrane patches, at negative and positive reference potentials, respectively. The results presented in this study demonstrate that membrane depolarization induces slow activation of stretch-activated channels of leech central neurons. This phenomenon is similar to that found in Xenopus oocytes, however, some peculiar features of the voltage dependence in leech stretch-activated channels indicate that specific membrane-glass interactions might not necessarily be involved. Moreover, following depolarization, stretch-activated channels in membrane patches from neurons in culture exhibited significantly shorter delay to activation (sec) than their counterparts from neurons of freshly isolated ganglia (hundreds of sec).

Painless Without ATP

The capsaicin receptor (CAP) is a cation channel involved in pain perception. Kwak et al. observed that when inside-out patches are prepared from dorsal root ganglia neurons for electrophysiological recording, the CAP activity is reduced, indicating that an intracellular regulator may be lost in the inside-out preparation. Adenosine trisphosphate (ATP) was found to enhance I CAP in response to application of capsaicin by a mechanism that did not require ATP hydrolysis and was not inhibited by treatments to block a variety of protein kinases. CAP has two putative nucleotide-binding domains and mutations in either of these domains eliminates the ability of ATP to enhance channel activity in response to capsaicin. The data suggest that ATP is an endogenous regulator of CAP. The K D for ATP to influence I CAP is within the range that would allow the ATP sites to be continuously occupied in vivo under normal conditions, but may be a mechanism for modulating pain perception during periods of cellular stress, such as ischemia. Kwak, J., Wang, M.H., Hwang, S.W., Kim, T.-Y., Lee, S.-Y., and Oh, U. (2000) Intracellular ATP increases capsaicin-activated channel activity by interacting with nucleotide binding domains. J. Neurosci. 20 : 8298-8304. [Abstract] [Full Text]

Painless Without ATP

The capsaicin receptor (CAP) is a cation channel involved in pain perception. Kwak et al. observed that when inside-out patches are prepared from dorsal root ganglia neurons for electrophysiological recording, the CAP activity is reduced, indicating that an intracellular regulator may be lost in the inside-out preparation. Adenosine trisphosphate (ATP) was found to enhance ICAP in response to application of capsaicin by a mechanism that did not require ATP hydrolysis and was not inhibited by treatments to block a variety of protein kinases. CAP has two putative nucleotide-binding domains and mutations in either of these domains eliminates the ability of ATP to enhance channel activity in response to capsaicin. The data suggest that ATP is an endogenous regulator of CAP. The KD for ATP to influence ICAP is within the range that would allow the ATP sites to be continuously occupied in vivo under normal conditions, but may be a mechanism for modulating pain perception during periods of cellular stress, such as ischemia.Kwak, J., Wang, M.H., Hwang, S.W., Kim, T.-Y., Lee, S.-Y., and Oh, U. (2000) Intracellular ATP increases capsaicin-activated channel activity by interacting with nucleotide binding domains. J. Neurosci. 20: 8298-8304. [Abstract] [Full Text]

Regulation of the Type III InsP 3 Receptor by InsP 3 and ATP

Many hormones and neurotransmitters raise intracellular calcium (Ca 2+) by generating InsP 3 and activating the inositol 1,4,5-trisphosphate receptor (InsP 3R). Multiple isoforms with distinct InsP 3 binding properties (Cardy et al., 1997) have been identified (Morgan et al., 1996). The type III InsP 3R lacks Ca 2+-dependent inhibition, a property that makes it ideal for signal initiation (Hagar et al., 1998). Regulation of the type III InsP 3R by InsP 3 and ATP was explored in detail using planar lipid bilayers. In comparison to the type I InsP 3R, the type III InsP 3R required a higher concentration of InsP 3 to reach maximal channel activity (EC 50 of 3.2 μM versus 0.5 μM for the types III and I InsP 3R, respectively). However, the type III InsP 3R did reach a 2.5-fold higher level of activity. Although activation by InsP 3 was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 μM InsP 3, low ATP concentrations (<6 mM) increased the open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature of type III InsP 3R regulation. Enhanced channel activity in the presence of high InsP 3 may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help to modulate intracellular Ca 2+ signaling.

Endogenous polyamines modulate Ca2+ channel activity in guinea-pig intestinal smooth muscle.

The polyamines spermine and spermidine inhibit L-type Ca2+ channels in whole-cell recordings from guinea-pig ileum cells (Gomez and Hellstrand, Pflügers Arch, 430:501-507, 1995 [4]). To study whether they modulate channel activity under physiological conditions, we further investigated their actions on Ca2+ channels and the effects of altered cellular polyamine contents. In inside-out patches, spermine (0.1-1 mM) inhibited channel activity without affecting the amplitude of unitary currents. In cell-attached recordings, addition of spermine to the bath did not influence channel activity in the patch, indicating that its extracellular action is direct and not mediated via passage of the polyamine through the cell membrane. Cellular contents of spermidine and spermine were decreased by about 50% by organ culture of ileum strips for 5 days with the adenosylmethionine decarboxylase inhibitor CGP 48664 (10 microM). This caused enhanced channel activity in cell-attached recordings, suggesting a reduced level of channel block by endogenous polyamines compared with control cells. Whole-cell recordings in the perforated patch mode showed increased current in polyamine-depleted cells, while this was not seen when cells were dialysed with the pipette solution. We conclude that polyamines block Ca2+ channels from the inside as well as the outside of the cell membrane, and that endogenous polyamines in smooth muscle modulate Ca2+ channel activity.

Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca 2+ Involves Luminal Ca 2+ Sensing Sites

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca 2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 μM to 2 mM Ca 2+ (free) and 3 mM ATP (total) on the cytosolic ( cis) side and 20 μM to 20 mM Ca 2+ on the luminal ( trans) side of the channel and with Cs + as the charge carrier. Under conditions of low trans Ca 2+ (20 μM), increasing cis Ca 2+ from 0.1 to 10 μM caused a gradual increase in channel open probability ( P o). Elevating cis Ca 2+ above 100 μM resulted in a gradual decrease in P o. Elevating trans [Ca 2+] enhanced channel activity (EC 50 ≈ 2.5 mM at 1 μM cis Ca 2+) primarily by increasing the frequency of channel openings. The dependency of P o on trans [Ca 2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca 2+ chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N, N-tetraacetic acid. Elevated luminal Ca 2+ enhanced the sensitivity of the channel to activating cytosolic Ca 2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca 2+. Potentiation of P o by increased luminal Ca 2+ occurred irrespective of whether the electrochemical gradient for Ca 2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca 2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca 2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca 2+ are mediated by distinct Ca 2+-sensitive site(s) at the luminal face of the channel or associated protein.

BK channels in intact clonal rat pituitary cells are activated by physiological elevations of the cytosolic Ca2+ concentration at the normal resting potential.

Activation of large conductance Ca(2+)-activated K+ channels (BK channels) in intact clonal rat pituitary cells (GH4 cells) was investigated using the cell-attached patch-clamp configuration. This method prevents loss of intracellular factors which might influence channel activity. BK channels are generally considered to be inactive at the resting membrane potential in excitable cells. However, at the resting potential (0 mV pipette potential), 40% of the cell-attached patches displayed spontaneously active BK channels, which remained active even at 20 mV hyperpolarization. The peptide thyroliberin (TRH) elevates the cytosolic Ca2+ concentration ([Ca2+]i) in GH cells by IP3-induced release of Ca2+ from intracellular stores. This rise in [Ca2+]i occurs concomitantly with membrane hyperpolarization. TRH stimulation caused activation of BK channels in nine out of 30 silent cell-attached patches, and caused enhanced channel activity in seven out of 29 cell-attached patches containing spontaneously active BK channels. The Ca2+ ionophore ionomycin activated silent BK channels in three out of 10 cell-attached patches, and increased the activity of spontaneously active BK channels in seven out of 16 cell-attached patches. The pipette potential was clamped to 0 mV in all these experiments. We conclude that the BK channels in GH4 cells may be active at the resting membrane potential and more negative membrane potentials. The channels may also be activated further by physiological elevations of [Ca2+]i in the same potential range. Our results point towards new possible physiological roles for the BK channels in GH4 cells. This is in agreement with the emerging picture of BK channels highly sensitive to [Ca2+]i in a wide variety of cell types.

The essential role of the Walker A motifs of SUR1 in K-ATP channel activation by Mg-ADP and diazoxide.

The ATP-sensitive K-channel (K-ATP channel) plays a key role in insulin secretion from pancreatic beta-cells. It is closed by glucose metabolism, which stimulates insulin secretion, and opened by the drug diazoxide, which inhibits insulin release. Metabolic regulation is mediated by changes in ATP and Mg-ADP, which inhibit and potentiate channel activity, respectively. The beta-cell K-ATP channel consists of a pore-forming subunit, Kir6.2, and a regulatory subunit, SUR1. We have mutated (independently or together) two lysine residues in the Walker A (W(A)) motifs of the first (K719A) and second (K1384M) nucleotide-binding domains (NBDs) of SUR1. These mutations are expected to inhibit nucleotide hydrolysis. Our results indicate that the W(A) lysine of NBD1 (but not NBD2) is essential for activation of K-ATP currents by diazoxide. The potentiatory effects of Mg-ADP required the presence of the W(A) lysines in both NBDs. Mutant currents were slightly more sensitive to ATP than wild-type currents. Metabolic inhibition led to activation of wild-type and K1384M currents, but not K719A or K719A/K1384M currents, suggesting that there may be a factor in addition to ATP and ADP which regulates K-ATP channel activity.

Modulation of inwardly rectifying ATP-regulated K+ channel by phosphorylation process in opossum kidney cells.

The role of phosphorylation in modulating an inwardly rectifying ATP-regulated K+ channel with inward conductance of about 90 pS was examined using the patch-clamp technique on opossum kidney (OK) cells. The activity of the inwardly rectifying K+ channel observed in cell-attached patches rapidly declined (channel "rundown") upon excision of the membrane into inside-out patches in a control bath solution (3 mM Mg2+, ATP-free). The declined channel activity was partially restored by applying ATP to the bath, and the ATP-induced channel restoration reached the near maximal level at an ATP concentration of 3 mM. The channel activity maintained by 3 mM ATP in inside-out patches was inhibited by K-252a (10 microM), a nonspecific protein kinase inhibitor, or KT5720 (200 nm), a specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA), and was further stimulated by the addition of a catalytic subunit of PKA (20 nM). In cell-attached patches, the channel activity was also inhibited by K-252a (10 microM) or KT5720 (200 nM). The application of dibutyryl-cAMP (100 microM) alone failed to enhance channel activity, but significantly stimulated channel activity after the pretreatment of cells with Ro-20-1724 (100 microM), an inhibitor of cAMP-specific phosphodiesterase. These results suggest that maintenance of the activity of ATP-regulated K+ channels in OK cells requires protein kinase-mediated phosphorylation with ATP-hydrolysis, and that phosphorylation is mainly induced by PKA.

Activation of Plant Plasma Membrane Ca2+-Permeable Channels by Race-Specific Fungal Elicitors.

The response of plant cells to invading pathogens is regulated by fluctuations in cytosolic Ca2+ levels that are mediated by Ca2+-permeable channels located at the plasma membrane of the host cell. The mechanisms by which fungal elicitors can induce Ca2+ uptake by the host cell were examined by the application of conventional patch-clamp techniques. Whole-cell and single-channel experiments on tomato (Lycopersicon esculentum L.) protoplasts revealed a race-specific fungal elicitor-induced activation of a plasma membrane Ca2+-permeable channel. The presence of the fungal elicitor resulted in a greater probability of channel opening. Guanosine 5[prime]-[[beta]-thio]diphosphate, a GDP analog that locks heterotrimeric G-proteins into their inactivated state, abolished the channel activation induced by the fungal elicitor, whereas guanosine 5[prime][[gamma]-thio]triphosphate, a nonhydrolyzable GTP analog that locks heterotrimeric G-proteins into their activated state, produced an effect similar to that observed with the fungal elicitor. Mastoparan, which stimulates GTPase activity, mimicked the effect of GTP[[gamma]]S. The addition of HA1004 (a protein kinase inhibitor) in the presence of the elicitor totally abolished channel activity, whereas okadaic acid (a protein phosphatase inhibitor) moderately enhanced channel activity, suggesting that the activation of the channel by fungal elicitors is modulated by a heterotrimeric G-protein-dependent phosphorylation of the channel protein.

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Identification of a PY motif in the epithelial Na channel subunits as a target sequence for mutations causing channel activation found in Liddle syndrome.

Liddle syndrome is an autosomal dominant form of hypertension, resulting from mutations in the cytoplasmic C-terminus of either the beta or gamma subunits of the amiloride-sensitive epithelial Na channel (ENaC) which lead to constitutively increased channel activity. Most mutations reported to date result in the elimination of 45-75 normal amino acids from these segments, leaving open the question of the identity of the precise amino acids in which mutation can lead to an enhanced channel activity. To address this question, we have performed a systematic mutagenesis study of the C-termini of the alpha, beta and gamma ENaC subunits of the rat channel and have analyzed their function by expression in Xenopus oocytes. The results demonstrate that a short proline-rich segment present in the cytoplasmic C-terminus of each subunit is required for the normal regulation of channel activity. Missense mutations altering a consensus PPPXY sequence of the alpha, beta or gamma subunits reproduced the increase in channel activity found in mutants in which the entire cytoplasmic C-termini are deleted. This proline-rich sequence, referred to as the PY motif, is known to be a site of binding by proteins bearing a WW domain. These findings show that the three PY motifs in the C-termini of ENaC are involved in the regulation of channel activity, probably via protein-protein interactions. This new regulatory mechanism of channel function is critical for the maintenance of normal Na reabsorption in the kidney and of Na+ balance and blood pressure.