Wolfgang Zeiske

Ba2+-induced conductance fluctuations of spontaneously fluctuating K+ channels in the apical membrane of frog skin (Rana temporaria)

SummaryWe studied the influence of mucosal Ba2+ ions on the recently described (Zeiske & Van Driessche, 1979a, J. Membrane Biol.47:77) transepithelial, mucosa towards serosa directed K+ transport in the skin ofRana temporaria. The transport parametersG (conductance), PD (potential difference),Isc (short-circuit current, “K+ current”), as well as the noise ofIsc were recorded. Addition of millimolar concentrations of Ba2+ to the mucosal K+-containing solution resulted in a sudden but quickly reversible drop inIsc.G andIsc decreased continuously with increasing Ba2+ concentration, (Ba2+)o. The apparent Michaelis constant of the inhibition by Ba2+ lies within the range 40–80 μm. The apical membrane seems to remain permselective for K+ up to 500 μm (Ba2+)o. Higher (Ba2+)o, however, appears to induce a shunt (PD falls,G increases). This finding made an accurate determination of the nature of the inhibition difficult but our results tend to suggest a K+-channel block by K+−Ba2+ competition. In the presence of Ba2+, the power spectrum of the K+ current shows a second Lorentzian component in the low-frequency range, in addition to the high-frequency Lorentzian caused by spontaneous K+-channel fluctuations (Van Driessche & Zeiske, 1980). Both Lorentzian components are only present with mucosal K+ and can be depressed by addition of Cs+ ions, thus indicating that Ba2+ ions induce K+-channel fluctuations. The dependence of the parameters of the induced Lorentzian on (Ba2+)o, shows a rise in the plateau values to a maximum around 60 μm (Ba2+)o, followed by a sharp and progressive decrease to very low values. The corner frequency which reflects the rate of the Ba2+-induced fluctuations, however, increases quasi-linearly up to 1mm (Ba2+)o with a tendency to saturate at higher (Ba2+)o. Based on a three-state model for the K+ channel (having one open state, one closed by the spontaneous fluctuation and one blocked by Ba2+) computer calculations compared favorably with our results. The effect of Ba2+ could be explained by assuming reversible binding at the outer side of the apical K+ channel, thereby blocking the open channel in competition with K+. The association-dissociation of Ba2+ at its receptor site is thought to cause a chopping of the K+ current, resulting in modulated current fluctuations.

Ca2+-sensitive, spontaneously fluctuating, cation channels in the apical membrane of the adult frog skin epithelium

The fluctuations in transepithelial current through the abdominal skin of bullfrogs (Rana catesbeiana) were analysed while the transepithelial voltage was clamped to zero. A Lorentzian component in the power spectrum was recorded when the skin was bathed with Ca2+ free NaCl Ringers on both sides. After replacement of all mucosal Na+ by choline the Lorentzian component disappeared. The application of mucosa positive potentials enhanced the plateau of the relaxation noise component while it was depressed by mucosa negative potentials. These observations showed that the current associated with the relaxation noise, was carried by Na+ moving in the inward direction. Divalent cations added to the mucosal solution in micromolar concentrations depressed the relaxation noise immediately, which is indicative for an apical localization of the fluctuating channels. The relaxation noise depended strongly on the pH of the mucosal medium: alkalinization enhanced the relaxation noise while acidification depressed the fluctuations. Micromolar concentrations of the diuretic amiloride, which is known to block the Na+ entry into the cellular compartment, enhanced the Na+-dependent relaxation noise while at higher concentrations an inhibitory effect was observed. From these observations it was concluded that the relaxation noise is caused by inward Na+ movement through fluctuating channels which are localized in the apical membrane. These channels seem to constitute a pathway in parallel with the amiloride-blockable channels. Ionic substitution of Na+ by other monovalent cations showed that these channels are also permeable for K+, Rb+, NH4+, Cs+ and Tl+, but not for Li+. Divalent cations in micromolar concentrations completely occlude these fluctuating channels. Therefore, this pathway will be blocked for monovalents cations when normal Ca2+ containing Ringers are used as mucosal bathing medium.

Noise analysis reveals K+ channel conductance fluctuations in the apical membrane of rabbit colon.

SummaryIn this paper we describe current fluctuations in the mammalian epithelium, rabbit descending colon. Pieces of isolated colon epithelium bathed in Na+ or K+ Ringers solutions were studied under short-circuit conditions with the current noise spectra recorded over the range of 1–200 Hz. When the epithelium was bathed on both sides with Na+ Ringers solution (the mucosal solution contained 50 μm amiloride), no Lorentzian components were found in the power spectrum. After imposition of a potassium gradient across the epithelium by replacement of the mucosal solution by K+ Ringers (containing 50 μm amiloride), a Lorentzian component appeared with an average corner frequency,fc=15.6±0.91 Hz and a mean plateau valueSo=(7.04±2.94)×10−20 A2 sec/cm2. The Lorentzian component was enhanced by voltage clamping the colon in a direction favorable for K+ entry across the apical membrane. Elimination of the K+ gradient by bathing the colon on both sides with K+ Ringers solutions abolished the noise signal. The Lorentzian component was also depressed by mucosal addition of Cs+ or tetraethylammonium (TEA) and by serosal addition of Ba2+. The one-sided action of these K+ channel blockers suggests a cellular location for the fluctuating channels. Addition of nystatin to the mucosal solution abolished the Lorentzian component. Serosal nystatin did not affect the Lorentzian noise. This finding indicates an apical membrane location for the fluctuating channels. The data were similar in some respects to K+ channel fluctuations recorded from the apical membranes of amphibian epithelia such as the frog skin and toad gallbladder. The results are relevant to recent reports concerning transcellular potassium secretion in the colon and indicate that the colon possesses spontaneously fluctuating potassium channels in its apical membranes in parallel to the Na+ transport pathway.

Na+ channels and amiloride-induced noise in the mammalian colon epithelium

(1) The effects of the Na+-channel blocker, amiloride, on the short-circuit current carried by Na+ was studied with fluctuation analysis, in rabbit descending colon epithelium. (2) In the presence of mucosal amiloride, the power spectrum of the Na+-current noise showed a Lorentzian component. When the Na+ current was reduced by increasing the blocker concentrations, the Lorentzian plateau decreased and corner frequency increased. Macroscopic short-circuit current and current-noise data are evidence for a two-state mechanism of the blocker interaction with the Na+ channel. (3) On- and off-rate constants for the blocker-receptor reaction, single-channel currents and Na+-channel density were calculated at room temperature and at 37 degrees C. Also, the activation energy for the amiloride-receptor reaction was estimated. The microscopic parameters obtained for the Na+ channel in the colon were similar to those found for Na+ channels in other tight epithelia.

Saturable K+ pathway across the outer border of frog skin (rana temporaria): kinetics and inhibition by Cs+ and other cations.

SummaryThe reaction of abdominal skins of the frog speciesRana temporaria on mucosal K+-containing solutions was studied in an Ussing-type chamber by recording transepithelial potential difference (PD), short-circuit current (SCC) and conductance (G). With Na-Ringers as serosal medium, a linear correlation between PD and the logarithm of the mucosal K+-concentration ([K]o) was obtained. The K+-dependent SCC saturated with increasing [K]o, and could quickly and reversibly be depressed by addition of Rb+, Cs+, and H+, Li+, Na+, and NH4+ did not influence K+ current. A large scatter was obtained for kinetic parameters like the slope of the PD-log [K]o-line (18–36.5 mV/decade), the apparent Michaelis constant (13–200mm), and the maximal current of the saturable SCC (6–50 μA·cm−2), as well as for the degree of inhibition by Cs+ ions. This seemed to be caused by a time-dependent change during long time exposure to high [K]o (more than 30 sec), thereby inducing a selectivity loss of K+-transporting structures, together with an increase in SCC andG and a decrease in PD. Short time exposure to K+-containing solutions showed a competitive inhibition of K+ current by Cs+ ions, and a Michaelis constant of 6.6mm for the inhibitory action of Cs+. Proton titration resulted in a decrease of K+ current at pH<3. An acidic membrane component (apparent dissociation constant 2.5×10−3m) is virtually controlling K+ transfer. Reducing the transepithelial K+-concentration gradient by raising the serosal potassium concentration was accompanied by the disappearance of SCC and PD.

Current-noise analysis of the basolateral route for K+ ions across a K+-secreting insect midgut epithelium (Manduca sexta)

Abstract1.The isolated midgut of a lepidopteran larva (Manduca sexta), 5th instar was investigated with voltageclamp and fluctuation analysis techniques.2.With high K+ insect saline on both sides the outward-directed short-circuit current (Isc) was carried by K+ (IK) from serosal to mucosal compartment.IK could be blocked, in a dose-dependent manner by serosal Ba2+ ions. There was no current with serosal Na+.3.Noise analysis ofIK revealed a Lorentzian component in the power spectrum when Ba2+ was present in the serosal solution. The Ba2+/receptor kinetics show pseudo-first order characteristics only at low [Ba2+]s. For [Ba2+]s>KBa, the apparrent Ba2+ association rate decreases with a hyperbolic course as a function of serosal [Ba2+] which could indicate some “substrate-inhibition”-like interaction of Ba2+ at its receptor site.4.It is concluded that the serosal membranes of the K+-secreting intestinal cells contain the common type of Ba2+-blockable K+ channel which provides the serosal pathway for K+ during secretion which is ultimately driven by the mucosally-located electrogenic K+-ATPase.

The interaction of «K+-like» cations with the apical K+ channel in frog skin

SummaryThe apparent permeability of the apical K+ channel in the abdominal skin of the frog (Rana temporaria) for different monovalent cations was tested by comparing the shortcircuit current (SCC) obtained after imposition of serosally directed ionic concentration gradients. Furthermore, the SCC was subjected to noise analysis. Of various cations tested, only the “K+-like” ions NH4+, Rb+ and Tl+, besides K+, were found to permeate the apical K+ channel, as reflected by SCC- and fluctuation analysis: (i) The SCC could be depressed by addition of the K+-channel blocker Ba2+ to the mucosal solution. (ii) With the K+-like ions (Ringers concentration), a spontaneous Lorentzian noise was observed. Plateau values were similar for K+ and Tl+, and smaller for NH4+ and Rb+. The corner frequencies clearly increased in the order K+<NH4+<Tl+≪Rb+. The SCC dose-response relationships revealed a Michaelis-Menten-type current saturation only for pure K+- or Tl+-Ringers solutions as mucosal medium, whereas a more complicated SCC behavior was seen with Rb+ and especially, NH4+. For K+-Tl+ mixtures an anomalous mole-fraction relationship was observed: At low [Tl+]/[K+] ratios, Tl+ ions appeared to inhibit competitively the K+ current while, at high [Tl+]/[K+] ratios, Tl+ seemed to be a permeant cation. This feature was also detected in the noise analysis of K+−Tl+ mixtures. Long-term exposure to mucosal Tl+ resulted in an irreversible deterioration of the tissue. The SCC depression by Ba2+ was of a simple saturation-type characteristic with, however, different half-maximal doses (NH4+<K+<Rb+). Ba2+ induced a “blocker noise” in presence of all permeant cations with corner frequencies that depended on the Ba2+ concentration. A linear increase of the corner frequencies of the Ba2+-induced noise with increasing Ba2+ concentration was seen for NH4+, Rb+ and K+. With the assumption of a pseudo two-state model for the Ba2+ blockade the on- and off-rate constants for the Ba2+ interaction with the NH4+/Rb+/K+ channel were calculated and showed marked differences, dependent on the nature of the permeant ion. The specific problems with Tl+ prevented such an analysis but SCC- and noise data indicated a comparably poor efficiency of Ba2+ as Tl+-current inhibitor. We attempted a qualitative analysis of our results in terms of a “two-sites, three-barriers” model of the apical K+ channel in frog skin.

Poorly selective cation channels in the skin of the larval frog (Stage≤XIX)

The abdominal skin of bullfrog larvae (Rana catesbeiana) was placed in an Ussing-type chamber, and its transepithelial electrical parameters were recorded with mucosal solutions of different ionic composition. With “K+-like” cations (K+, NH4+, Rb+, Cs−) the power spectra of the fluctuations in short-circuit current displayed a Lorentzian component (fc=30–40 Hz). The relaxation noise could be suppressed by addition of the K+-channel blockers Ba2+ and TEA to the mucosal solution. Also, in presence of the ionophore antibiotic nystatin the Lorentzian noise was abolished. The Na+-channel probes amiloride and benzimidazolyl-2-guanidine (BIG) both enhanced the relaxation noise obtained with the K+-like cations but, with Na+, and Li+, also caused the rise of a relaxation component above the background noise. In presence of amiloride or BIG, the addition of Ba2+, TEA and nystatin still abolished the Lorentzian noise. It can be concluded that the relaxation-noise source is located in the apical cell membranes of the tadpole skin. These spontaneously fluctuating cation channels do not seem to strictly discriminate between K+-like ions (K+, NH4+, Rb+, Cs+) and Na+-like ions (Na+, Li+). On the other hand, well-known specific probes for K+ channels (Ba2+, TEA) and for Na+ channels (amiloride, BIG) interact with this apical cation channel. It is possible that the poorly selective channel plays a role in the ontogenesis of the specific Na+ transport in the maturing frog skin.

Secretory apical Cl– channels in A6 cells: possible control by cell Ca2+ and cAMP

Abstract Distal kidney cells (A6) from Xenopus laevis were cultured to confluency on porous supports. Tissues were mounted in Ussing-type chambers to measure short-circuit current (Isc), transepithelial conductance and capacitance, and to analyse the fluctuation in Isc. In the absence of apical NaCl, but with normal basolateral NaCl Ringer’s solution, extracellular addition of ATP, oxytocin, a membrane-permeant cAMP derivative, and forskolin produced a transient increase of the electrical parameters. Noise analysis revealed a spontaneous Lorentzian component. All responses depend strictly on the presence of basolateral Cl– and are caused by the activation of an apical (CFTR type) Cl– permeability. Repetitive treatment with ATP (or oxytocin) resulted in refractoriness. ATP and oxytocin acted antagonistically, whereas cAMP and ATP had additive effects. Incubation with the vesicular Ca2+ pump inhibitor thapsigargin or application of the Ca2+ channel blocker nifedipine elicited finite but variable Cl– channel activity. After treatment with nifedipine or thapsigargin, the response to oxytocin was severely impaired. We speculate that not only cAMP but also cell Ca2+ plays a crucial role in the activation of CFTR in A6. Ca2+ may be multifunctional but the rise in capacitance (apical area) observed with all stimulants strongly suggests its involvement in, and contribution to, exocytosis in the process of the CFTR-mediated transcellular Cl– movements.

Apical K+ channels in frog skin (Rana temporaria): Cation adsorption and voltage influence gating kinetics

Open-close kinetics of fluctuating K+ channels in the apical frog skin membrane were studied with noise analysis of the K+ current (IK). The mucosa to serosa directedIK was obtained with serosal NaCl-and mucosal KCl-Ringer under voltage clamp conditions. Mucosal protons (pH>4), several polyvalent metal ions, and choline shifted the plateaus (S0) of the Lorentzian component in theIK noise spectrum to higher, but the corner frequency (fc) to lower values.S0 was lowered at pH<4, due to a K+-channel block by H+. Ca2+, Sr2+, H+ (pH>4) and choline did not affectIK. A slight reduction ofIK was seen with Mg2+, Mn2+, Co2+, Ni2+, Zn2+, Cu2+ and La3+. At pH>4, the H+-induced shifts inSo anfc were almost abolished in solutions of high mucosal Ca2+ concentrations. Clamping the transepithelial potential difference to more positive values (with respect to the serosa) shifted the Lorentzian parametersS0 andfc in the same way as the cations did. As with protons, mucosal Ca2+ interferred with the effect of voltage. The interference of cationic (probably fixed charge screening) and voltage effects suggests a common, more general mechanism of action, namely alterations in K+-channel fluctuation kinetics by changes in local electrical fields. On this basis, the rates for the open-close reaction of K+ channels and their mean lifetime were calculated. We found that e.g. increasing [Ca2+]0 from 1–10 mM caused no change of the mean open time, but increased the mean time “closed” of the K+ channel by a factor of about 1.5. Other mucosal cations, as well as depolarizing clamp potentials are thought to have the same effect.