and stimulation by IBMX did not change the ionomycin induced Ca2+ increase in Xenopus oocytes. Taken togeth- er, these results suggest that activation of ...
Pflügers Arch – Eur J Physiol (1997) 435:178–181
© Springer-Verlag 1997
S H O R T C O M M U N I C AT I O N
&roles:K. Kunzelmann · M. Mall · M. Briel · A. Hipper R. Nitschke · S. Ricken · R. Greger
The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl– conductance of Xenopus oocytes
&misc:Received: 17 June 1997 / Received after revision: 4 September 1997 / Accepted: 5 September 1997
&p.1:Abstract Oocytes from Xenopus laevis activate a Ca2+ dependent Cl– conductance when exposed to the Ca2+ ionophore ionomycin. This Ca2+ activated Cl– conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (GI– / GCl–) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl– conductance, which produces more linear I/V curves with a GI– / GCl– ratio of about 0.52. Ionomycin enhanced CaCC (∆G) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (µS) 23 ± 1.9 (n=9) and 23.6 ± 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 ± 0.48 µS (n=18) and 6.9 ± 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. ∆F508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl– channel in oocytes inhibited CaCC to a lesser degree (∆G=12.1 ± 1.1 µS; n=7). Changes of CFTR and CaCCCl– whole cell conductances were also measured when extracellular Cl– was replaced by I–. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca2+. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca2+ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl– currents is paralleled by an inhibition of Ca2+ activated Cl– currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl– conductance. They might explain previous findings in epithelial tissues of CF-knockout mice. K. Kunzelmann (✉) · M. Mall · M. Briel · A. Hipper · R. Nitschke S. Ricken · R. Greger Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany&/fn-block:
& ey words CFTR · Ca2+ · Chloride channels · kwd:K Ionomycin · Xenopus oocytes · CF&bdy:
Introduction Oocytes of Xenopus laevis endogenously express Ca2+ activated Cl– currents (CaCC) [5]. These channels contribute to the baseline conductance measured in resting oocytes and are activated by increase of intracellular Ca2+. This conductance is outwardly rectified and has a halide selectivity ratio of I– > Cl–. Both characteristics have been frequently used to discriminate CaCC from other Cl– conductances like that produced by the cystic fibrosis transmembrane conductance regulator (CFTR) [4, 13]. According to previous reports CaCC and CFTRCl– conductance apparently are caused by independent proteins [4]. However, in studies with human colonic epithelial cells (HT29) we found that both CaCC and CFTR-Cl– currents were not additive [8] These and other findings [15] prompted the question of whether both conductances co-operate. Along these lines, it turned out only recently that CFTR can act as a regulator of other ion channels [11, 12, 15]. The CFTR-dependent inhibition of the epithelial Na+ channel (ENaC) has been studied in [11, 16]. These new findings and the studies described above prompted us to perform additional studies examining a possible interaction of CFTR with the endogenous CaCC in Xenopus oocytes. In contrast to previously described experiments with HT29 cells the properties of endogenous CaCC in Xenopus oocytes are significantly different from those of CFTR. The data presented here suggest inhibition of CaCC by activation of CFTR.
Materials and methods Preparation of cRNA for wild-type CFTR and ∆F508-CFTR. &p.2:A 4.7 kb cDNA sequences encoding wild type (wt) CFTR and ∆F508-CFTR (the most common CFTR mutation caused by deletion of Phe at position 508 were subcloned into p-Bluescript vec-
179 tor (Stratagene) using the restriction sites KpnI and NotI and amplified in E. coli (XL1-Blue, Stratagene). For in vitro transcription of cRNA the plasmids was linearized with KpnI and cRNA was synthesised using T7 promoter with the respective polymerase and a 5’ cap (mCAP mRNA capping kit, Stratagene). Preparation of oocytes and microinjection of cRNA. &p.2:Isolation and microinjection of oocytes have been described in a previous report [11]. In brief, after isolation from adult Xenopus laevis female frogs (H. Kähler, Bedarf für Entwicklungsbiologie, Hamburg, Germany), oocytes were dispersed and defolliculated by a 1 h-treatment with collagenase (type A, Boehringer, Germany). Subsequently, oocytes were rinsed 10 times and kept in ND96-buffer (in mmol/l): NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5, Na-pyruvate 2.5, pH 7.55), supplemented with theophylline (0.5 mmol/l) and gentamycin (5 mg/l) at 18°C. Oocytes of identical batches were injected each with 10–50 ng cRNA (wt CFTR, G551DCFTR) dissolved in about 50 nl double-distilled water (PV830 pneumatic pico pump, WPI, Germany). Oocytes injected with 50 nl double-distilled water served as controls. Electrophysiological analysis of Xenopus oocytes. &p.2:2–4 days after injection oocytes were impaled with two electrodes (Clark instruments) which had resistances of ≤1 MΩ when filled with 2.7 mol/l KCl. A flowing (2.7 mol/l) KCl electrode served as bath reference. The membrane currents were measured by voltage clamping of oocytes (OOC-1, WPI, Germany) from –60 to +40 mV in steps of 10 mV. The conductances were obtained from complete I/V curves. Here we report the chord conductances calculated for positive currents (reversal potential (=ECFTR≈ECACC) – +40 mV) according to Ohm’s law. This range was chosen because it describes the anion influx in the anion replacement studies. Measurement of intracellular cAMP. &p.2:Oocytes were incubated with IBMX for 10 min which was sufficient for complete activation of wtCFTR and ∆F508 (steady state activation). Half of the oocytes were exposed subsequently to ionomycin (1 µmol/l) for one min. Afterwards ooyctes were washed with ND96 solution and subsequently lysed in 70% ice cold ethanol. Lysates of two oocytes were pooled and cAMP was measured using an enzyme linked assay (cAMP Biotrack enzyme immuno assay system, Amersham). Materials. &p.2:All used compounds were of highest available grade of purity. 3-isobutyl-1-methylxanthine (IBMX), 8-(4-Chlorophenylthio)-adenosine 3’,5’-cyclic mono-phosphate (cpt-cAMP) and ionomycin were obtained from Sigma (Deisenhofen, Germany). Statistical analysis was performed according to Students t test. P values
