The glycine residues G551 and G1349 within the ATP-binding cassette signature motifs play critical roles in the activation and inhibition of cystic fibrosis transmembrane conductance regulator channels by phloxine B

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.     

Comparative Pharmacology of the Activity of Wild-type and G551D Mutated CFTR Chloride Channel: Effect of the Benzimidazolone Derivative NS004

The pharmacological activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel mutated at glycine 551 (G551D-CFTR) was studied in the presence of the benzimidazolone derivative NS004 and compared to that of wild-type (wt) CFTR. Using iodide (¹²⁵I) efflux and whole-cell patch-clamp techniques we found dose-dependent stimulation of phosphorylated wt-CFTR channels by NS004 with an EC ₅₀ 11 µM. With non-phosphorylated CFTR, the effect of NS004 was apparent only at concentration >100 µM. In G551D-CFTR-expressing CHO cells, neither forskolin (from 0.1 to 10 µM) nor NS004 (from 0.1 to 200 µM) added separately were able to stimulate channel activity. However, in the presence of 10 µM forskolin, NS004 stimulated G551D-CFTR activity in a dose-dependent manner with an EC ₅₀ 1.5 µM. We also determined the half-maximal effective concentration of forskolin (EC ₅₀ 3.2 µM) required to stimulate G551D channel activity in presence of 1.5 µM NS004. No inhibitory effect was observed at high concentration of NS004 with both wt- and G551D-CFTR. Whole-cell recordings of CFTR chloride currents from cells expressing wild-type or G551D-CFTR in the presence of NS004 were linear, time- and voltage-independent. The inhibitory profile of G551D-CFTR channel activity was similar to that of wild type, i.e., inhibition by glibenclamide (100 µM) and DPC (250 µM) but not by DIDS (200 µM) nor calixarene (100 nM). These results show that NS004 activates wt-CFTR channel and restores G551D-CFTR channel activity, the potency of which depends on both the concentration of NS004 and the phosphorylation status of CFTR.     

Differential sensitivity of the cystic fibrosis (CF)-associated mutants G551D and G1349D to potentiators of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel

The genetic disease cystic fibrosis (CF) is caused by loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Two CF mutants, G551D and G1349D, affect equivalent residues in the highly conserved LSGGQ motifs that are essential components of the ATP-binding sites of CFTR. Both mutants severely disrupt CFTR channel gating by decreasing mean burst duration (MBD) and prolonging greatly the interburst interval (IBI). To identify small molecules that rescue the gating defects of G551D- and G1349D-CFTR and understand better how these agents work, we used the patch clamp technique to study the effects on G551D- and G1349D-CFTR of phloxine B, pyrophosphate (PP(i)), and 2'-deoxy ATP (2'-dATP), three agents that strongly enhance CFTR channel gating. Phloxine B (5 microm) potentiated robustly G551D-CFTR Cl- channels by altering both MBD and IBI. In contrast, phloxine B (5 microm) decreased the IBI of G1349D-CFTR, but this effect was insufficient to rescue G1349D-CFTR channel gating. PP(i) (5 mm) potentiated weakly G551D-CFTR and was without effect on the G1349D-CFTR Cl- channel. However, by altering both MBD and IBI, albeit with different efficacies, 2'-dATP (1 mm) potentiated both G551D- and G1349D-CFTR Cl- channels. Using the ATP-driven nucleotide-binding domain dimerization model of CFTR channel gating, we suggest that phloxine B, PP(i) and 2'-dATP alter channel gating by distinct mechanisms. We conclude that G551D- and G1349D-CFTR have distinct pharmacological profiles and speculate that drug therapy for CF is likely to be mutation-specific.     

G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects

Mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR) result in cystic fibrosis (CF). CFTR is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis at its nucleotide binding domains (NBDs). G551D-CFTR, the third most common CF-associated mutation, has been characterized as having a lower open probability (Po) than wild-type (WT) channels. Patients carrying the G551D mutation present a severe clinical phenotype. On the other hand, G1349D, also a mutant with gating dysfunction, is associated with a milder clinical phenotype. Residues G551 and G1349 are located at equivalent positions in the highly conserved signature sequence of each NBD. The physiological importance of these residues lies in the fact that the signature sequence of one NBD and the Walker A and B motifs from the other NBD form the ATP-binding pocket (ABP1 and ABP2, named after the location of the Walker A motif) once the two NBDs dimerize. Our studies show distinct gating characteristics for these mutants. The G551D mutation completely eliminates the ability of ATP to increase the channel activity, and the observed activity is approximately 100-fold smaller than WT-CFTR. G551D-CFTR does not respond to ADP, AMP-PNP, or changes in [Mg(2+)]. The low activity of G551D-CFTR likely represents the rare ATP-independent gating events seen with WT channels long after the removal of ATP. G1349D-CFTR maintains ATP dependence, albeit with a Po approximately 10-fold lower than WT. Interestingly, compared to WT results, the ATP dose-response relationship of G1349D-CFTR is less steep and shows a higher apparent affinity for ATP. G1349D data could be well described by a gating model that predicts that binding of ATP at ABP1 hinders channel opening. Thus, our data provide a quantitative explanation at the single-channel level for different phenotypes presented by patients carrying these two mutations. In addition, these results support the idea that CFTR's two ABPs play distinct functional roles in gating.     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Confers Glibenclamide Sensitivity to Outwardly Rectifying Chloride Channel (ORCC) in Hi-5 Insect Cells

Increasing evidence is now accumulating for the involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) in the control of the outwardly rectifying chloride channel (ORCC). We have examined the sensitivity of ORCC to the sulfonylurea drug glibenclamide in Hi-5 (Trichoplusia ni) insect cells infected with recombinant baculovirus expressing either wild-type CFTR, ΔF508-CFTR or E. coliβ galactosidase cDNA and in control cells either infected with virus alone or uninfected. Iodide efflux and single channel patch-clamp experiments confirmed that forskolin and 1-methyl-3-isobutyl xanthine (IBMX) or 7-methyl-1,3 dipropyl xanthine (DPMX) activate CFTR channels (unitary conductance: 9.1 ± 1.6 pS) only in cells expressing CFTR. In contrast, we identified 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS)-sensitive ORCC in excised membrane patches in any of the cells studied, with similar conductance (22 ± 2.5 pS at −80 mV; 55 ± 4.1 pS at +80 mV) and properties. In the presence of 500 μm SITS, channel open probability (P _o ) of ORCC was reversibly reduced to 0.05 ± 0.01 in CFTR-cells, to 0.07 ± 0.02 in non-CFTR expressing cells and to 0.05 ± 0.02 in ΔF508-cells. In Hi-5 cells that did not express CFTR, glibenclamide failed to inhibit ORCC activity even at high concentrations (100 μm), whereas 500 μm SITS reversibly inhibited ORCC. In contrast in cells expressing CFTR or ΔF508, glibenclamide dose dependently (IC₅₀= 17 μm, Hill coefficient 1.2) and reversibly inhibited ORCC. Cytoplasmic application of 100 μm glibenclamide reversibly reduced P _o from 0.88 ± 0.03 to 0.09 ± 0.02 (wash: P _o = 0.85 ± 0.1) in CFTR cells and from 0.89 ± 0.05 to 0.08 ± 0.05 (wash: P _o = 0.87 ± 0.1) in ΔF508 cells. In non-CFTR expressing cells, glibenclamide (100 μm) was without effect on P _o (control: P _o = 0.89 ± 0.09, glib.: P _o = 0.86 ± 0.02; wash: P _o = 0.87 ± 0.05). These data strongly suggest that the expression of CFTR confers glibenclamide sensitivity to the ORCC in Hi-5 cells. Received: 23 October 1998/Revised: 29 December 1998     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996     

Evidence for Negative Charge in the Conduction Pathway of the Cardiac Ryanodine Receptor Channel Provided by the Interaction of K+ Channel N-type Inactivation Peptides

We have investigated the interaction of two peptides (ShB — net charge +3 and ShB:E12KD13K — net charge +7) derived from the NH₂-terminal domain of the Shaker K⁺ channel with purified, ryanodine-modified, cardiac Ca²⁺-release channels (RyR). Both peptides produced well resolved blocking events from the cytosolic face of the channel. At a holding potential of +60 mV the relationship between the probability of block and peptide concentration was described by a single-site binding scheme with 50% saturation occurring at 5.92 ± 1.06 μm for ShB and 0.59 ± 0.14 nm for ShB:E12KD13K. The association rates of both peptides varied with concentration (4.0 ± 0.4 sec⁻¹μm ⁻¹ for ShB and 2000 ± 200 sec⁻¹μm ⁻¹ for ShB:E12KD13K); dissociation rates were independent of concentration. The interaction of both peptides was influenced by applied potential with the bulk of the voltage-dependence residing in K_off. The effectiveness of the inactivation peptides as blockers of RyR is enhanced by an increase in net positive charge. As is the case with inactivation and block of K⁺ channels, this is mediated by a large increase in K_on. These observations are consistent with the proposal that the conduction pathway of RyR contains negatively charged sites which will contribute to the ion handling properties of this channel. Received: 15 December 1997/Revised: 13 March 1998     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation

We have designed and synthesizedbenzo[c]quinolizinium derivatives and evaluated their effects on theactivity of G551D cystic fibrosis transmembrane conductance regulator(CFTR) expressed in Chinese hamster ovary and Fisher ratthyroid cells. We demonstrated, using iodide efflux, whole cell patchclamp, and short-circuit recordings, that5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91)restored the activity of G551D CFTR (EC₅₀ = 85 µM)and activated CFTR in Calu-3 cells (EC₅₀ = 47 µM).MPB-91 has no effect on the ATPase activity of wild-type and G551DNBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylatekinase activities of purified NBD2. The activation of CFTR by MPB-91 isindependent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTRhaving 10 mutated protein kinase A sites (10SA-CFTR). The newpharmacological agent MPB-91 may be an important candidate drug toameliorate the ion transport defect associated with CF and to point outa new pathway to modulate CFTR activity.

     

Apical Heterotrimeric G-proteins Activate CFTR in the Native Sweat Duct

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl⁻ conductance (CFTR G _Cl ) in the native sweat duct (SD). We permeabilized the basolateral membrane with α-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G _Cl activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-γ-S (100 μm) also activates CFTR G _Cl in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-γ-S increased CFTR G _Cl by 44 ± 20 mS/cm² (mean ±se; n= 7). GDP (10 mm) inhibited G-protein activation of CFTR G _Cl even in the presence of GTP-γ-S. The heterotrimeric G-protein activator (AlF₄ ⁻) in the cytoplasmic bath activated CFTR G _Cl (increased by 51.5 ± 9.4 mS/cm² in the presence of 5 mm ATP without cAMP, n= 6), the magnitude of which was similar to that induced by GTP-γ-S. Employing immunocytochemical-labeling techniques, we localized Gαs, Gαi, Gαq, and Gβ at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G _Cl in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G _Cl activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G _Cl activity. Received: 9 June 2000/Revised: 5 October 2000     

Competitive inhibition by substrates of the esterification reaction between l-phenylalanine and d-glucose catalysed by the lipases of Rhizomucor miehei and Candida rugosa

A kinetic study on esterification between d-glucose and l-phenylalanine catalysed by lipases from Rhizomucor miehei (RML) and Candida rugosa (CRL) in organic media investigated in detail showed that both the lipases followed a Ping-Pong Bi-Bi mechanism with two distinct types of competitive inhibitions. Graphical double reciprocal plots and computer simulation studies showed that competitive double substrate inhibition took place at higher concentrations leading to dead-end inhibition in the case of RML and in the case of CRL, inhibition only by d-glucose at higher concentrations leading to dead-end lipase–d-glucose complexes. An attempt to obtain the best fit of these kinetic models through curve-fitting yielded in good approximation, the apparent values of important kinetic parameters, RML: k _cat = 2.24 ± 0.23 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 95.6 ± 9.7 mM, K _{m d-glucose} = 80.0 ± 8.5 mM, K _{i l-phenylalanine} = 90.0 ± 9.2 mM, K _{i d-glucose} = 13.6 ± 1.42 mM; CRL: k _cat = 0.51 ± 0.06 mM h⁻¹ (mg protein)⁻¹, K _{m l-phenylalanine} = 10.0 ± 0.98 mM, K _{m d-glucose} = 6.0 ± 0.64 mM, K _{i d-glucose} = 8.5 ± 0.81 mM.     

Membrane Potassium Channels and Human Bladder Tumor Cells. I. Electrical Properties

These experiments were conducted to determine the membrane K⁺ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K⁺ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca²⁺-dependent outward K⁺ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca²⁺-activated, voltage-dependent K⁺ channel (K_Ca) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K_Ca activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K⁺ channel, 21 ± 1 pS at positive transmembrane potential (V _m ) and 145 ± 13 pS at negative V _m . Glibenclamide (50 μm), Ba²⁺ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K⁺ channels in inside/out patches in a dose-dependent manner, and the IC₅₀= 46 μm. The identity of this K⁺ channel with an ATP-sensitive K⁺ channel (K_ATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the K_Ca and a lower conductance K⁺ channel with properties consistent with a sulfonylurea receptor-linked K_ATP. Received: 12 June 1997/Revised: 21 October 1997     

A pull-down assay for 5' AMP-activated protein kinase activity using the GST-fused protein

An assay using a specific peptide (SAMS peptide) as a substrate is widely used for determination of AMP-activated protein kinases (AMPK) activity. However, it is not an efficient assay for crude AMPK preparations. In this study, we modified the assay by using the SAMS peptide fused to glutathione-S-transferase (GST-SAMS) instead of the SAMS peptide on its own. Radioactivity incorporated into GST-SAMS can be recovered easily by precipitation with glutathione-agarose. The kinetic parameters of partially purified AMPK for the GST-SAMS were as follows. The V_max was 0.26±0.012 nmol/min/mg of total proteins and K _m for GST-SAMS was 110±12 μM. The parameters for ATP were 0.40±0.016 nmol/min/mg of total proteins (V_max) and 202±21 μM (K _m ). The activity of AMPK in this system was stimulated about threefold by the AMPK activators, AMP or 5-amino-4-imidazolecarboxamide ribotide (ZMP), and inhibited by the AMPK inhibitors, adenine 9-β-d-arabinofuranoside (ara-A) and iodotubercidin. These values correlate well with those for the SAMS peptide reported previously. Thus, we succcssfully established a convenient and rapid method to measure AMPK applicable, even for crude enzyme preparations.     

Purification and Kinetic Properties of 6-Phosphogluconate Dehydrogenase from Rat Small Intestine

6-Phosphogluconate dehydrogenase (6PG) was purified from rat small intestine with 36% yield and a specific activity of 15 U/mg. On SDS/PAGE, one band with a mass of 52 kDa was found. On native PAGE three protein and two activity bands were observed. The pH optimum was 7.35. Using Arrhenius plots, Ea, ΔH, Q₁₀ and Tm for 6PGD were found to be 7.52 kcal/mol, 6.90 kcal/mol, 1.49 and 49.4°C, respectively. The enzyme obeyed “Rapid Equilibrium Random Bi Bi” kinetic model with K_m values of 595 ± 213 μM for 6PG and 53.03±1.99 μM for NADP. 1/V_m versus 1/6PG and 1/NADP plots gave a V_m value of 8.91±1.92 U/mg protein. NADPH is the competitive inhibitor with a K_i of 31.91±1.31 μM. The relatively small K_i for the 6PGD:NADPH complex indicates the importance of NADPH in the regulation of the pentose phosphate pathway through G6PD and 6PGD.     

Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

The patch-clamp technique was used to investigate the effects ofthe isoflavone genistein on disease-causing mutations (G551D andF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing thetrafficking-competent G551D-CFTR, the forskolin-stimulated Cl currentswere small, and average open probability of G551D-CFTR wasP_o = 0.047 ± 0.019. Addition of genistein activated Cl currents~10-fold, and the P_o of G551D-CFTRincreased to 0.49 ± 0.12, which is aP_o similar towild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous forthe trafficking-impaired F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These datasuggested that genistein activated CFTR mutants that were present inthe cell membrane. Therefore, we tested the effects of genistein in CFpatients with the G551D mutation in nasal potential difference (PD)measurements in vivo. The perfusion of the nasal mucosa of G551D CFpatients with isoproterenol had no effect; however, genisteinstimulated Cl-dependent nasal PD by, on average, 2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to -adrenergicstimulation) found in healthy subjects.

     

Characterization and Distribution of a Cloned Rat μ-Opioid Receptor

Abstract: We have cloned and expressed a rat brain cDNA, TS11, that encodes a μ-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [³H]diprenorphine with high affinity (K_D = 0.23 ± 0.04 nM). The rank order potency of drugs competing with [³H]diprenorphine was as follows: levorphanol (K_i = 0.6 ± 0.2 nM) ≈β-endorphin (K_i = 0.7 ± 0.5 nM) ≈ morphine (K_i = 0.8 ± 0.5 nM) ≈ [d -Ala², N-Me-Phe⁴,Gly-ol⁵]-enkephalin (DAMGO; K_i = 1.6 ± 0.5 nM) ? U50,488 (K_i = 910 ± 0.78 nM) > [d -Pen^2,5]-enkephalin (K_i = 3,170 ± 98 nM) > dextrorphan (K_i = 4,100 ± 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar K_i are consistent with a μ-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) In COS-7 cells 50 µM 5′-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC₅₀ = 3.4 ± 0.5 nM) to a lower-affinity state (IC₅₀ = 89.0 ± 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned μ-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the μ-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of μ-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.     

ATP hydrolysis by a CFTR domain: pharmacology and effects of G551D mutation

Residues 417-830 of the cystic fibrosis transmembrane conductance regulator (CFTR) were expressed as a glutathione-S-transferase fusion protein. This fusion protein, NBD1/R/GST, contains the regulatory and first nucleotide binding domains of CFTR. NBD1/R/GST hydrolyzed ATP with a K(M) (60 microM) and V(max) (330 nmol/min/mg) that differed from those reported for CFTR and for a peptide containing CFTR residues 433-589. The ATPase inhibitor profile of NBD1/R/GST indicates that CFTR resembles P-glycoprotein with respect to the NBD1 ATPase catalytic mechanism. ATP hydrolysis by NBD1/R/GST was unaffected by genistein, glybenclamide, and other agents known to affect CFTR's chloride channel function, suggesting that these agents do not act by directly influencing the ATPase function of NBD1. The disease-causing mutation, G551D, reduced ATP hydrolysis by NBD1/R/GST by increasing the K(M) for ATP fourfold. This suggests that when G551D occurs in patients with cystic fibrosis, it affects CFTR function by reducing the affinity of NBD1 for ATP.     

Identification of a Region Critically Involved in the Interaction of Phlorizin with the Rabbit Sodium-D-Glucose Cotransporter SGLT1

In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604–610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of α-methyl-d-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K _i for phlorizin increased at least by a factor of 5 compared to the wild-type K _i of 4.6 ± 0.7 μmol/l; most striking changes were observed for Y604K (K _i = 75.3 ± 19.0 μmol/l) and C608K (K _i = 83.6 ± 13.9 μmol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (K _m = 0.8 ± 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside. Received: 29 March 2001/Revised: 15 June 2001