Wolf-Michael Weber

Expression Cloning of a Cdna From Rabbit Small-Intestine Related to Proton-Coupled Transport of Peptides, Beta-Lactam Antibiotics and Ace-Inhibitors

Injection of poly(A)+ RNA from rabbit small intestine intoXenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino ß-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H+-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in ooyctes clamped at a holding potential of-60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

Abstract. The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.

Influence of extracellur Ca2+ on endogenous Cl− channels in Xenopus oocytes

Removal of Ca2+ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl− efflux, which could be strongly inhibited by the Cl− channel blockers flufenamic acid and niflumic acid. Removal of Mg2+ or Ba2+ from the solutions had the same effects as removing Ca2+. The reversal potential of −12 mV also indicated that Cl− channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca2+ concentration of about 20 μM showed that quite low doses of extracellular Ca2+ profoundly influence the electrical properties of the oocyte membrane.

Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.

1 To gain information on the mode of operation of the renal proton‐coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2 Using differently charged glycyl‐dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH‐dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2–50 μm) between pH 5.5 and 7.4 and membrane potentials of ±0 to −50 mV. 3 Maximal substrate‐evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pH0). Potential‐dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4 The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from –50 to –150 mV substrate affinities decrease 10‐ to 50‐fold whereas apparent Imax increases almost 10‐fold. 5 Even at saturating H+/H3O+ and dipeptide concentrations the I–V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6 These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl− channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately −15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl−. Readdition of Ca2+ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca2+ inactivated Cl− channel (CaIC). The inhibitory potency of Ca2+ was a function of the external Ca2+ concentration with a half maximal blocker concentration of about 20 μm.These channels were inhibited by the Cl− channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4′-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonicacid (SITS), another Cl− channel blocker, led to activation of this Cl− channel. Like other Cl− channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl− current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl− channels in Xenopus oocytes which share several features with Cl− channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl− channels.

Amiloride-sensitive Na+ conductance in native Xenopus oocytes

Endogenous Na+ conductances in the plasma membrane of oocytes of the South African clawed toad Xenopus laevis were investigated by microelectrode techniques and influx measurements. Removal of Na+ from the bath solution under voltage clamp conditions led to a decrease in the clamp current indicating the existence of native Na+ conductances. The observed current was voltage dependent but showed no marked rectification. Amiloride (10 microM) blocked this Na+ current reversibly. However, amiloride analogues such as benzamil and phenamil had no effect on this Na+ conductance. The Na+/H(+)-exchanger blocker EIPA (5-(N-ethyl-N-isopropyl)amiloride), another amiloride analogue, also had no effect thereby excluding a possible involvement of the Na+/H+ exchanger. The Na+ mediated current had a reversal potential of about 50 mV suggesting high selectivity of these Na+ conductances for Na+ over other monovalent cations. When Na+ was replaced by K+ in the bath solution, amiloride had no effect on the clamp current over the whole potential range demonstrating that only Na+ but not K+ can enter the cell via the investigated conductances. In radio tracer experiments 22Na+ influx into oocytes was nearly halved in presence of amiloride (10 microM), whereas benzamil and phenamil again failed to influence 22Na+ influx. These results suggest that the endogenous amiloride-sensitive Na+ conductance belongs to a new class of channels which is quite different from amiloride-sensitive epithelial Na+ channels.

cAMP sensitivity conferred to the epithelial Na+ channel by α-subunit cloned from guinea-pig colon

Abstract. The rate of Na+ (re)absorption across tight epithelia such as in distal kidney nephron and colon is to a large extent controlled at the level of the epithelial Na+ channel (ENaC). In kidney, antidiuretic hormone (ADH, vasopressin) stimulates the expression/activity of this channel by a cAMP/protein-kinase-A- (PKA-) mediated pathway. However, a clear upregulation of ENaC function by cAMP could not be reproduced with cloned channel subunits in the Xenopus oocyte expression system, suggesting the hypothesis that an additional factor is missing. In contrast, we show here that membrane-permeant cAMP can activate ENaC expressed in Xenopus oocytes (3.8-fold) upon replacement of the rat α-subunit by a new α-subunit cloned from guinea-pig colon (gpα). This α-subunit is 76% identical with its rat orthologue originating from ADH-insensitive rat colon. The biophysical fingerprints of the hybrid ENaC formed by this guinea-pig α-subunit together with rat β- and γ-subunits are indistinguishable from those of rat ENaC (rENaC). Injection of the PKA inhibitor PKI-(6–22)-amide into the oocyte had no effect on the basal activity of rat ENaC but inhibited the activity of gpα-containing hybrid ENaC and greatly decreased its stimulation by cAMP. This suggests that, unlike for rat ENaC, tonic PKA activity is required for basal function of gpα-containing ENaC and that PKA mediates its cAMP-induced activation. This regulatory behaviour is not common to all ENaCs containing an α-subunit cloned from an ADH-responsive tissue since xENaC, which was cloned from the ADH-sensitive Xenopuslaevis A6 epithelia, is, when expressed in oocytes, resistant to cAMP, similar to rat ENaC. This study demonstrates that the PKA sensitivity of ENaC can depend on the nature of the ENaC α-subunit and raises the possibility that cAMP can stimulate ENaCs by different mechanisms.

Stretch-independent activation of the mechanosensitive cation channel in oocytes of Xenopus laevis

Oocytes of the South African clawed toad Xenopus laevis possess in their plasma membrane a so-called stretch-activated cation channel (SAC) which is activated by gently applying positive or negative pressure (stretch) to the membrane patch containing the channels. We show here that this mechanosensitive channel acted as a spontaneously opening, stretch-independent non-selective cation channel (NSCC) in more than half of the oocytes that we investigated. In 55% of cell-attached patches (total number of patches, 58) on 30 oocytes from several different donors, we found NSCC opening events. These currents were increased by elevating the membrane voltage or raising the temperature. NSCC and SAC currents shared some properties regarding the relative conductances of Na+>Li+>Ca2+, gating behaviour and amiloride sensitivity. Stretch-independent currents could be clearly distinguished from stretch induced SAC currents by their voltage and temperature dependence. Open events of NSCC increased strongly when temperature was raised from 21 to 27 degrees C. NSCC currents could be partly inhibited by high concentrations of extracellular Gd3+ and amiloride (100 and 500 microM, respectively). We further show exemplarily that NSCC can seriously hamper investigations when oocytes are used for the expression of foreign ion channels. In particular, NSCC complicated investigations on cation channels with small conductance as we demonstrate for a 4 pS epithelial Na+ channel (ENaC) from guinea pig distal colon. Our studies on NSCCs suggest the involvement of these channels in oocyte temperature response and ion transport regulation. From our results we suggest that NSCC and SAC currents are carried by one protein operating in different modes.

Effects of nicotine on human nasal epithelium: evidence for nicotinic receptors in non-excitable cells

Abstract We investigated the effects of nicotine and its derivate nicotine di-d-tartrate on primary cultured human nasal epithelial cells. Both substances evoked an in-crease in the intracellular free calcium concentration. In the presence of extracellular Ca2+ the cytosolic Ca2+ ([Ca2+]i) increase was long lasting, whereas in the absence of external Ca2+ there was a transient increase of [Ca2+]i indicating that nicotine has an influence on Ca2+ conductances across the membranes and on intracellular Ca2+ stores. Both effects could be blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). Apical or basolateral application of nicotine during transepithelial transport measurements with confluent monolayers of cultured human nasal cells resulted in a significant, reversible decrease of amiloride-sensitive sodium absorption with an apparent half-maximal blocker concentration of about 950 μM. To exclude the possibility that remnant neuronal components were responsible for the observed effects we used tetrodotoxin and verapamil to block putative neuronal channels and 4-(4-diethyla- mino)styryl-N-methylpyridinium iodide (4-di-2-Asp) to stain neuronal tissue. Both experimental approaches demonstrated that there were no neuronal-mediated effects. These results indicate the direct effects of nicotine on human nasal epithelium, giving the first evidence of the existence of nicotinic receptors in non-excitable cells.

Effects of Benzamil in Human Cystic Fibrosis Airway Epithelium

Cystic fibrosis airway epithelia exhibit a defective transepithelial electrolyte transport. The cyclic adenosine monophosphate stimulated Cl– secretion is abolished because of the loss of t