Electrophysiology of the parietal cell.
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Amphibians↗
Biomedical subjects
D D Loo
Publications and source records attributed to D D Loo.
Activation of apical chloride channels in the gastric oxyntic cell.
Oxyntic cells that retain distinct morphological polarity between apical and basolateral membranes were isolated from the gastric mucosa of the amphibian Necturus. Patch-clamp techniques were applied to these cells to identify apical membrane ion channels associated with hydrochloric acid secretion. A single class of voltage-dependent, inwardly rectifying chloride channels was observed in the apical membranes of both resting and stimulated (acid-secreting) oxyntic cells. Stimulation of the cells with dibutyryladenosine 3',5'-monophosphate and isobutylmethylxanthine increased channel open probability and simultaneously increased apical membrane surface area. This chloride channel is probably responsible for electrogenic chloride secretion by the gastric mucosa and may also participate in the fluid- and enzyme-secretory functions of the oxyntic cell, analogous to the chloride channels found in the apical membranes of other exocrine cells.
1-Methyl-3-isobutylxanthine↗
Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon.
Using patch-clamp techniques, we have studied Ca2+-activated K+ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately -45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131 +/- 25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K+ over Na+. Channel activity correlated directly with bath Ca2+ concentration in the excised patches. Channel currents were blocked by 5 mM TEA+ and 1 mM Ba2+. In cell-attached patches, after addition of the Ca2+ ionophore A23187, which increases intracellular Ca2+, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1 mM dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca2+. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca2+ and cAMP.
Animals↗
Differential modulation of Ca2(+)-activated K+ channels by substance P.
Substance P released from motoneurons or primary afferent nerves innervating the gut is an excitatory noncholinergic regulator of gastrointestinal motility and has been shown to stimulate contractions in longitudinal colonic muscle. By using the patch-clamp technique on single myocytes from the longitudinal muscle of the rabbit colon, we have studied the action of the peptide on membrane conductances. In cell-attached patches, addition of the peptide to the bath activates a large conductance Ca2(+)-activated K+ channel. At peptide concentrations (10(-12) M) that did not result in cell contraction, K+ channels were activated in a synchronized, cyclical fashion. The activation did not occur in the presence of nifedipine (10(-6) M), a blocker of dihydropyridine-sensitive Ca2+ channels. The activation was also absent when the cells were depolarized in 126 mM KCl-Ringer solution. In contrast, at a concentration of the peptide (10(-7) M) that resulted in cell contraction, there was a transient activation of K+ channels, followed by a prolonged inhibition. Nifedipine (10(-6) M) did not block the K+ channel activation by this concentration of the peptide. Removal of bath Ca2+ abolished activation of the K+ channels by both the high and low concentrations of substance P. These results indicate that substance P exerts its effect via two different membrane pathways for extracellular Ca2+.
Animals↗
Ca2+-activated K+ channels in the apical membrane of Necturus choroid plexus.
The properties of Ca2+-activated K+ channels in the apical membrane of the Necturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K+ over Na+ and K+ over Cs+. They were blocked by high cytoplasmic Na+ concentrations (110 mM). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca2+ concentrations. Increasing Ca2+ from 5 to 500 nM, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca2+ was 500 nM. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba2+ reduced both channel-open probability and conductance. The sites for the action of Ba2+ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117 mM were calculated as the apparent Ba2+ dissociation constants (KD(0 mV] for the effects on probability and conductance, respectively. TEA+ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with a KD(0 mV) = 5.6 mM. A second site, with a higher affinity, KD(0 mV) = 0.23 mM, may account for the near total block of channel conductance by 2 mM TEA+ applied to the outside of the membrane. It is concluded that the channels in Necturus choroid plexus exhibit many of the properties of "maxi" Ca2+-activated K+ channels found in other tissues.
Animals↗
Ca2+-activated K+ currents in Necturus choroid plexus.
The tight-seal whole-cell recording method has been used to study Necturus choroid plexus epithelium. A cell potential of -59 +/- 2 mV and a whole cell resistance of 56 +/- 6 M omega were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110 mM NaCl Ringer solution and the interior of the cell contained a solution of 110 mM KCl and 5 nM Ca2+, stepping the membrane potential from a holding value of -50 to -10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca2+-activated K+ channels. Based on the voltage dependence of the activation of Ca2+-activated K+ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K+ as their reversal potential at different K+ concentration gradients followed the Nernst potential for K+. These currents were reduced by the addition of TEA+ to the bath solution and were eliminated when Cs+ or Na+ replaced intracellular K+. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t1/2) to the steady value. Increasing the pipette Ca2+ also decreased the delay and decreased t1/2. For instance, when pipette Ca2+ was increased from 5 to 500 nM, the delay and t1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K+ currents through Ca2+-activated K+ channels. At the resting membrane potential of -60 mV, Ca2+-activated K+ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20-30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca2+. Thus the Ca2+-activated K+ channels may play a specific role in maintaining intracellular K+ concentrations during CSF secretion.
Animals↗
Regulation of K+ channels in the basolateral membrane of Necturus oxyntic cells.
Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands from Necturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67 +/- 21 pS in 24% of the patches and channels of smaller conductance, 33 +/- 6 pS in 56% of the patches. Channels in both classes were highly selective for K+ over Na+ and Cl-, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca2+ in the range between 5 and 500 nM, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca2+. The presence of K+ channels in the basolateral membrane which are regulated by these known "second messengers" can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.
Animals↗
Crystal accumulation and very high short-circuit currents in rabbit urinary bladder.
We describe a condition in rabbits characterized by CaCO3 crystal accumulation and very high short-circuit current (Isc) in the urinary bladder. The incidence of the condition was high in rabbits kept for 2 mo or more in the vivarium. The crystal mass in the bladder increased with age or time until it occupied up to one-third of the bladder volume and occasionally aggregated as stones, and the urine attained a sludgy consistency. In animals with sludgy urine, the urine excreted had a crystal content 20 times lower than that of urine contained in the bladder at time of death, implying retention of crystals in the bladder. Kidney stones were not detected in rabbits with bladder crystals, and bladder crystals were absent in rabbits with kidney stones induced experimentally by uremia. In old rabbits with sludgy urine the Isc, a measure of Na+ transport, was up to 46 microA/cm2 and averaged 12 microA/cm2, seven times the Isc of rabbits with nonsludgy urine. The increased Isc was entirely amiloride sensitive. Noise analysis showed it to arise entirely from increased channel density, without change in single-channel current. With one possible exception, we could not find bacterial infection or abnormalities in plasma aldosterone, GFR, or urinary [Na+], [Ca2+], pH, or osmolality that could explain the condition. The exception is that some unidentified component accounting for half the osmolality of nonsludgy urine is absent or at low concentration in sludgy urine. It remains unknown why the condition develops and whether CaCO3 crystals cause high Isc or vice versa or whether both result from a third factor.
Amiloride↗
Studies of sodium channels in rabbit urinary bladder by noise analysis.
Sodium channels in rabbit urinary bladder were studied by noise analysis. There are two components of short-circuit current (Isc) and correspondingly two components of apical Na+ entry, one amiloride-sensitive (termed IA and the A channel, respectively) and one amiloride-insensitive (IL and the leak pathway, respectively). The leak pathway gives rise to l/f noise, while the A channel in the presence of amiloride gives rise to Lorentzian noise. A two-state model of the A channel accounts well for how the corner frequency and plateau value of Lorentzian noise vary with amiloride concentration. The single-channel current is 0.64 pA, and the conducting channel density is on the order of 40 copies per cell. Triamterene blocks the A channel alone, and increasing external Na+ decreases the number but not the single-channel permeability of the A channel. Hydrostatic pressure pulses ("punching") increase the number of both pathways. Repeated washing of the mucosal surface removes most of the leak pathway without affecting the A channel. Properties of the A channel revealed by noise analysis of various tight epithelia are compared, and the mechanism of l/f noise is discussed. It is suggested that the A channel is synthesized intracellularly, stored in intracellular vesicles, transferred with or from vesicular membrane into apical membrane under the action of microfilaments, and degraded into the leak pathway, which is washed out into urine or destroyed. The A channel starts with PNa/PK approximately 30 and loses selectivity in stages until PNa/PK reaches the free-solution mobility ratio (approximately 0.7) for the leak pathway. This turnover cycle functions as a mechanism of repair and regulation for Na+ channels, analogous to the repair and regulation of most intracellular proteins by turnover. Vesicular delivery of membrane channels may be operating in several other epithelia.
Amiloride↗
Turnover, membrane insertion, and degradation of sodium channels in rabbit urinary bladder.
Noise analysis of rabbit bladder revealed two components: Lorentzian noise, arising from interaction of amiloride with the Na+ channel, and flicker noise (l/f, where f is frequency), as in other biological membranes. Hydrostatic pressure, which causes exchange between intracellular vesicular membrane and apical membrane, increases the number but not the single-channel current of the amiloride-sensitive channels. Flicker noise arises from degraded channels that have lost amiloride sensitivity and Na+ to K+ selectivity. The degraded channels were selectively removed by washing the mucosal surface. These results imply channel turnover by intracellular synthesis, transfer from vesicular to apical membrane, degradation, and elimination.
Amiloride↗
Some observations on the behaviour of chloride current--voltage relations in Xenopus muscle membrane in acid solutions.
Chloride current--voltage relations in Xenopus laevis muscle membrane have been investigated in phosphate-buffered solution (pH 5.2--5.4) using a three-microelectrode voltage clamp. Resting chloride conductance in these conditions is about 10(-4) S/cm2, approximately 1/10th that at pH 8.8. When the membrane potential is stepped from the holding (resting) potential to a more negative voltage the current rises from the initial to the steady state. The instantaneous current--voltage relation is linear and the steady-state relation shows inward-going rectification. As hyperpolarization appears to "activate" the chloride conductance, the "availability" of chloride current has been measured at the beginning of a voltage step to a standard test potential following conditioning at a variety of potentials. The relationship between the test current and the conditioning voltage is sigmoid. The normalized sigmoid curve has the same slope (absolute value) but opposite sign to that obtained in the same experiment at pH 8.8. In mildly acidic solutions (pH 6.4) the current wave form is diphasic: current initially falls then rises to the steady state. This combination of transients militates against the idea that transients are due solely to accumulation--depletion effects in restricted spaces ("unstirred layers") and a hypothesis is qualitatively outlined in which pH-and voltage-dependent effects are ascribed to a single type of channel whose orientation in the membrane is unconstrained.
Acids↗
Voltage dependence of chloride current through Xenopus muscle membrane in alkaline solutions.
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Alkalies↗