1. Whole cell voltage-clamp recordings were made from GH3 cells, a clonal cell line initially derived from a rat anterior pituitary tumor, using patch electrodes filled with CsCl or N-methylglucamine chloride (NMG Cl). The bathing medium contained tetraethylammonium chloride (TEA; 20 mM) and NaCl (120 mM) or NMG Cl (140 mM). These conditions resulted in substantial blockade of outward currents. 2. Depolarizing voltage steps from a holding potential of -50 mV activated transient (T-type) and sustained (L-type) inward Ca2+ currents. In addition, prolonged depolarization (>1 s) invariably elicited a slowly activating inward current that persisted with maintained depolarization, and deactivated slowly on repolarization, resulting in a prominent inward tail current. 3. This tail current could be recorded under conditions where Ca2+ and Cl- were the only membrane-permeant ions (symmetrical NMG Cl). The tail current nulled near 0 mV with symmetrical Cl- and showed a negative reversal potential with nominally Cl--free internal solution. Ba2+ substituted for Ca2+ as a carrier of inward current, but no tail current was expressed. These observations indicate that Cl- is the charge carrier of the slow inward tail current. 4. The voltage dependence for activation of the slow tail current was U-shaped with a peak at ~ -10 mV. This closely paralleled the voltage dependency of the Ca2+ currents. Recordings with 5 mM internal ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetracetic acid (EGTA) to buffer intracellular Ca2+ to low nM levels exhibited slow inward tail currents that were of lower peak amplitude than with the usual 1.1 mM EGTA-containing pipette solution, but the kinetics of the currents were similar in both cases. In addition, the slow tail current was eliminated on superfusion with the Ca2+ channel blocker Cd2+ or with Ca2+ -free medium. These results demonstrate that the current is dependent on Ca2+ influx; it is, therefore, referred to as I(Cl(Ca)). 5. Activation of I(Cl(Ca)) required depolarization of at least 1 s. More prolonged depolarizations activated progressively greater current, to a maximum with 6-s depolarization. In most cases, the decay of the tail current was described by a single exponential function with time constant ~0.8-0.9 s within the potential range -80 to -30 mV. At more depolarized potentials the decay was slower (increasing e-fold/20-mV change in membrane potential). 6. In a high proportion of cells, I(Cl(Ca)) rapidly diminished in amplitude on repeated activation. This 'rundown' occurred more rapidly than the rundown of the Ca2+ currents. Addition of the nonhydrolyzable guanosine triphosphate (GTP) analogues 5'-guanylylimidodiphosphate (GppNHp; 100 μM) or guanosine 5'-O-(3-thiotriphosphate (GTPγS; 100 μM) slowed the rundown of both the Ca2+ and Cl- currents; however, there was a significantly greater effect on I(Cl(Ca)). GTP, ATP, 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP) or guanosine 5'-O-(2-thiodiphosphate) (GTPβS) did not prevent rundown. Neither pertussis toxin nor cholera toxin had a significant effect on the current or on the ability of the guanosine triphosphate (GTP) analogues to prevent rundown, suggesting that the effect of GTP analogues may not result from an interaction with pertussis or cholera toxin-sensitive GTP binding proteins. 7. These results demonstrate that clonal pituitary cells possess a slowly activating and deactivating Ca2+-dependent Cl- current [I(Cl(Ca))]. It is speculated that the current is only activated under circumstances of large transmembrane Ca2+ flux when intracellular Ca2+ levels rise to high levels due to an overwhelming of endogenous Ca2+ -buffering mechanisms. Alternatively, I(Cl(Ca)) could be activated on hormone-stimulated release of Ca2+ from intracellular binding sites. The rundown of the current may result from depletion of intracellular-free Ca2+, which can be partially prevented by the GTP analogues, possibly through an action on membrane phosphoinositide turnover.
|Original language||English (US)|
|Number of pages||17|
|Journal||Journal of Neurophysiology|
|State||Published - 1988|
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