The Journal of Membrane Biology

Strauß, O. ; Steinhausen, K. ; Mergler, S. ; Stumpff, F. ; Wiederholt, M.

doi: 10.1007/s002329900526pmid: 10354461

This combined study of patch-clamp and intracellular Ca2+ ([Ca2+] i ) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl− channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca2+] i and activation of Cl− currents. In contrast, intracellular application of Ca2+ (10 μm) only induced transient activation of Cl− currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of I CRAC, La3+ (10 μm), despite the fact that both maneuvers led to a decline in [Ca2+] i . The InsP3-induced rise in Cl− conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl−-conductance in 50% of the cells investigated without affecting [Ca2+] i . Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca2+] i and Cl− conductance. In summary, elevation of [Ca] i by InsP3 leads to activation of Cl− channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.

Blanchard, M.-P. ; Klauke, N. ; Zitzmann, S. ; Plattner, H.

doi: 10.1007/s002329900527pmid: 10354462

We analyzed [Ca2+] i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935–945; Plattner et al., 1994. J. Membrane Biol. 158:197–208). Wild-type cells (7S), nondischarge strain nd9–28°C and trichocyst-free strain ``trichless'' (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca2+] i transients with moderate [Ca2+] o in the culture/assay fluid (50 μm or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+] o = 30 nm (c.f. [Ca2+] rest i =∼50 to 100 nm), veratridine produced only a small cortical [Ca2+] i transient. This increased in size and spatial distribution at [Ca2+] o = 50 μm of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+] o = 10 mm, [Ca2+] i transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca2+] o = 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+] o = 50 μm to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+ o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may ``consume'' a large part of the [Ca2+] i increase. (iii) With unusually high [Ca2+] o , mobilization of cortical stores and/or Ca2+ o influx may be impeded by the known membrane stabilizing effect of Ca2+ o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein.

Lalevée, N. ; Joffre, M.

doi: 10.1007/s002329900528pmid: 10354463

We have characterized a Ca2+-dependent Cl− current (ClCa) in cultured Sertoli cells from immature rat testis by using the whole cell recording patch-clamp technique. Cells dialyzed with pipette solutions containing 3 mm adenoside-triphosphate (ATP) and 1 μm free Ca2+, exhibited outward currents which were inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid (9-AC) but insensitive to tetraethylammonium (TEA). Dialysis of cells with pipette solutions containing less than 1 nm free Ca2+ strongly reduced the currents indicating that they were Ca2+ dependent. With cells dialyzed with Cs+ glutamate-rich pipette solutions containing 0.2 mm EGTA, 10 μm ionomycin induced outward currents having properties of Ca2+-activated Cl− currents.

Chabot, H. ; Vives, M.F. ; Dagenais, A. ; Grygorczyk, Cz. ; Berthiaume, Y. ; Grygorczyk, R.

doi: 10.1007/s002329900529pmid: 10354464

Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl− into the cell and is negligible when Cl− flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (I amil ) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to −60 mV, stimulating CFTR with 1 mm IBMX reduced I amil by up to 80%, demonstrating that ENaC is inhibited when Cl− is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of I amil downregulation and the CFTR-mediated plasma membrane Cl− conductance, suggesting a direct correlation. However, I amil downregulation was not affected when Cl− flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl− reversal potential (67% ± 10% inhibition at −20 mV compared to 79% ± 4% at −60 mV) demonstrating that I amil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in I amil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit I amil . These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl− transport across the plasma membrane.

Tripathy, A. ; Xu, L. ; Pasek, D.A. ; Meissner, G.

doi: 10.1007/s002329900530pmid: 10354465

Single channel and [3H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca2+-activated (0.5–1 μm free Ca2+) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca2+ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC50≈ 2.5 mm. In agreement with single channel measurements, high-affinity [3H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca2+. BDM was without a noticeable effect at low (≤0.01 μm) Ca2+ concentrations. At 20 μm Ca2+, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca2+ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca2+ and tissue-dependent manner.

Takahashi, S. ; Santos-Sacchi, J.

doi: 10.1007/s002329900531pmid: 10354466

The underlying Boltzmann characteristics of motility-related gating currents of the outer hair cell (OHC) are predicted to generate distortion components in response to sinusoidal transmembrane voltages. We studied this distortion since it reflects the mechanical activity of the cell that may contribute to peripheral auditory system distortion. Distortion components in the OHC electrical response were analyzed using the whole-cell voltage clamp technique, under conditions where ionic conductances were blocked. Single or double-sinusoidal transmembrane voltage stimulation was delivered at various holding voltages, and distortion components of the current responses were detected by Fourier analysis. Current response magnitude and phase of each distortion component as a function of membrane potential were compared with characteristics of the voltage-dependent capacitance, obtained by voltage stair-step transient analysis or dual-frequency admittance analysis. The sum distortion was most prominent among the distortion components at all holding voltages. Notches in the sum (f1+f2), difference (f2−f1) and second harmonic (2f) components occur at the voltage where peak voltage-dependent capacitance resides (V pkCm ). Rapid phase reversals also occurred at V pkCm , but phase remained fairly stable at more depolarized and hyperpolarized potentials. Thus, it is possible to extract Boltzmann parameters of the motility-related charge movement from these distortion components. In fact, we have developed a technique to follow changes in the voltage dependence of OHC motility and charge movement by tracking the voltage at phase reversal of the f2−f1 product. When intracellular turgor pressure was changed, V pkCm and distortion notch voltages shifted in the same direction. These data have important implications for understanding cochlear nonlinearity, and more generally, indicate the usefulness of distortion analysis to study displacement currents.