A slow calcium-dependent chloride conductance in clonal anterior pituitary cells

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.