Currents In Primary Cultures Research Articles

Properties of single potassium channels in cultured primary astrocytes

The patch-clamp technique was used to characterize the single channel ion currents in primary cultures of rat astrocytes. The most dominant channel type, which was found in over half of the inside-out membrane patches, was a potassium channel. The measured reversal potential was −67 mV, which is close to the calculated Nernst potential ions (−80 mV). These potassium channels activated with burst of very brief openings. Once activated the channels did not inactivate. The measured probabilities of the channels to be closed showed at least 3 different modes of channel behaviour; one voltage-independent and two voltage-dependent modes. During each activity-mode a ‘main’ conductance level plus two other conductance levels were recorded. In some recordings a pronounced outward rectification could also be seen.

GTP-binding proteins mediate acetylcholine inhibition of voltage dependent calcium channels in hippocampal neurons

Acetylcholine (Ach) was tested for its effect on calcium currents in primary cultures of embryonic rat hippocampal neurones. 10 microM Ach reversibly depressed the high voltage activated (HVA) Ca current and the effect was blocked by atropine. When GTP binding proteins (G-proteins) were irreversibly activated by intracellular perfusing the neurons with GTP[S], one short-lasting application of Ach permanently inhibited Ca currents. Alternatively, the transmitter was inactive when the cells were preincubated with pertussis toxin, which inactivates some G-proteins by catalyzing ADP ribosylation. Our results suggest that a G-protein mediates the inhibitory modulation of the HVA Ca current by muscarinic agonists.

An Apical-Membrane Chloride Channel in Human Tracheal Epithelium

The mechanism of chloride transport by airway epithelia has been of substantial interest because airway and sweat gland-duct epithelia are chloride-impermeable in cystic fibrosis. The decreased chloride permeability prevents normal secretion by the airway epithelium, thereby interfering with mucociliary clearance and contributing to the morbidity and mortality of the disease. Because chloride secretion depends on and is regulated by chloride conductance in the apical cell membrane, the patch-clamp technique was used to directly examine single-channel currents in primary cultures of human tracheal epithelium. The cells contained an anion-selective channel that was not strongly voltage-gated or regulated by calcium in cell-free patches. The channel was also blocked by analogs of carboxylic acid that decrease apical chloride conductance in intact epithelia. When attached to the cell, the channel was activated by isoproterenol, although the channel was also observed to open spontaneously. However, in some cases, the channel was only observed after the patch was excised from the cell. These results suggest that this channel is responsible for the apical chloride conductance in airway epithelia.

Facilitation of Ca2+-channel currents in bovine adrenal chromaffin cells.

Ca2+-channel currents in primary cultures of bovine adrenal chromaffin cells were studied using the whole-cell patch-clamp method. Parameters of a double-pulse protocol were systematically varied to characterize facilitation by a prepulse (P1) of Ca2+-channel current during a test pulse (P2). The pulses were usually separated by 30 msec, an interval sufficient for decay of any measurable P1 tail currents. The Ca2+-channel current amplitude during P2 increased when P1 voltage was more positive than 0 mV. The effect became progressively greater with more positive P1 voltage. With a 60-msec P1 to +80 mV, the current amplitude typically increased by 25%-35% during a 60-msec P2. Comparison of facilitated and control inward Ca2+-channel current I(V) curves showed that facilitation was also strongly dependent on P2 test voltage. Facilitation of Ca2+-channel currents is a voltage-dependent phenomenon and is not dependent on Ca2+ entry. When short repetitive voltage-clamp pulses were applied, the Ca2+-channel current amplitude increased with each pulse. This suggests that Ca2+-channel facilitation could enhance release of catecholamines from chromaffin cells during a train of action potentials.