Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch recording.
The whole cell patch-clamp technique, in both standard and perforated patch configurations, was used to study the influence of Na+-Ca++ exchange on rundown of voltage-gated Ca++ currents and on the duration of tail currents mediated by Ca++-dependent Cl- channels. Ca++ currents were studied in GH3 pituitary cells; Ca++-dependent Cl- currents were studied in AtT-20 pituitary cells. Na+-Ca++ exchange was inhibited by substitution of tetraethylammonium (TEA+) or tetramethylammonium (TMA+) for extracellular Na+. Control experiments demonstrated that substitution of TEA+ for Na+ did not produce its effects via a direct interaction with Ca++-dependent Cl- channels or via blockade of Na+-H+ exchange. When studied with standard whole cell methods, Ca++ and Ca++-dependent Cl- currents ran down within 5-20 min. Rundown was accelerated by inhibition of Na+-Ca++ exchange. In contrast, the amplitude of both Ca++ and Ca++-dependent Cl- currents remained stable for 30-150 min when the perforated patch method was used. Inhibition of Na+-Ca++ exchange within the first 30 min of perforated patch recording did not cause rundown. The rate of Ca++-dependent Cl- current deactivation also remained stable for up to 70 min in perforated patch experiments, which suggests that endogenous Ca++ buffering mechanisms remained stable. The duration of Ca++-dependent Cl- currents was positively correlated with the amount of Ca++ influx through voltage-gated Ca++ channels, and was prolonged by inhibition of Na+-Ca++ exchange. The influence of Na+-Ca++ exchange on Cl- currents was greater for larger currents, which were produced by greater influx of Ca++. Regardless of Ca++ influx, however, the prolongation of Cl- tail currents that resulted from inhibition of Na+-Ca++ exchange was modest. Tail currents were prolonged within tens to hundreds of milliseconds of switching from Na+- to TEA+-containing bath solutions. After inhibition of Na+-Ca++ exchange, tail current decay kinetics remained complex. These data strongly suggest that in the intact cell, Na+-Ca++ exchange plays a direct but nonexclusive role in limiting the duration of Ca++-dependent membrane currents. In addition, these studies suggest that the perforated patch technique is a useful method for studying the regulation of functionally relevant Ca++ transients near the cytoplasmic surface of the plasma membrane.
Korn
S J
SJ
Neurosciences Department, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.
Horn
R
R
D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels; D002712:Chlorides; D004533:Egtazic Acid / Q000494:pharmacology; D004553:Electric Conductivity; D006706:Homeostasis; D007700:Kinetics; D008565:Membrane Proteins / Q000502:physiology*; D010903:Pituitary Gland, Anterior / Q000187:drug effects / Q000502:physiology*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D015220:Calcium Channels; D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; D013757:Tetraethylammonium Compounds; D004533:Egtazic Acid; D019789:Tetraethylammonium; D012964:Sodium; C027917:tetramethylammonium; D002118:Calcium