Aileen K. Ritchie

Tetraethylammonium blockade of apamin-sensitive and insensitive Ca2(+)-activated K+ channels in a pituitary cell line.

1. The pharmacological sensitivities and physiological contributions of two types of Ca2(+)‐activated K+ channels (BK and SK) in GH3 cells were examined by the outside‐out, whole‐cell and cell‐attached modes of the patch‐clamp technique. 2. BK channels (250‐300 pS in symmetrical 150 mM‐K+) in outside‐out patches were blocked by external tetraethylammonium (TEA) and by 50 nM‐charybdotoxin (CTX), but were not blocked by apamin. 3. SK channels (9‐14 pS in symmetrical 150 mM‐K+) in outside‐out patches were blocked by external TEA and by apamin, but were not blocked by 50 nM‐CTX. 4. The dissociation constant (Kd) for TEA block of SK channels (3.1 +/‐ 0.37 mM) was 12‐fold greater than the Kd for the BK channels (260 +/‐ 21 microM). The TEA blockade of both channels was not strongly voltage dependent: for both channels the TEA binding site sensed less than 20% of the membrane electric field. 5. Application of blockers of the BK channels (1 mM‐TEA and 50 nM‐CTX) to whole cells under current clamp prolonged action potential duration; whereas application of apamin, a selective blocker of the SK channel, inhibited a slowly decaying after‐hyperpolarization and had little effect on action potential duration. Apamin also increased the firing rate in 30% of the spontaneously pacing cells. 6. It is suggested that BK channels contribute to action potential repolarization: whereas SK channels contribute to the regulation of action potential firing rate.

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca2+ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca2+ action potentials but has a Ca2+ sensitivity at −50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca2+ at −50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca2+-activated K+ tails currents and during Ca2+-activated K+ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.

Two distinct calcium-activated potassium currents in a rat anterior pituitary cell line.

1. The single ‘giga‐seal’ patch‐electrode technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was used to record whole‐cell currents in the GH3 rat anterior pituitary cell line. 2. GH3 cells have a rapidly inactivating, voltage‐dependent K+ current that is selectively inhibited by 4‐aminopyridine (4‐AP) but not by tetraethylammonium chloride (TEA). 3. The majority of the Ca2+‐activated K+ current in these cells is blocked by TEA with an inhibitory concentration that is half‐maximal at 1 mM. An additional Ca2+‐activated K+ current is also present that is relatively resistant to TEA and is blocked by the polypeptide apamin. The apamin‐sensitive component represents less than 18% of the total Ca2+‐activated K+ current at 0 mV. 4. The time course of the slowly declining components of the Ca2+‐activated K+ tail currents measured at the ‐50 mV holding potential was usually biexponential with time constants of 0.21 +/‐ 0.02 and 1.75 +/‐ 0.23 s (mean +/‐ S.E. of mean, n = 14). Both of the two slowly decaying components contribute to the TEA‐ and apamin‐sensitive currents. 5. It is concluded that GH3 cells have at least two pharmacologically distinct Ca2+‐activated K+ currents and a 4‐AP‐sensitive voltage‐dependent K+ current.

Corticotropin releasing hormone inhibits an inwardly rectifying potassium current in rat corticotropes.

1 The perforated‐patch‐clamp technique was used to identify an inwardly rectifying K+ current (IK(IR)) in cultured rat anterior pituitary cells highly enriched in corticotropes. IK(IR) was rapidly activating and highly selective for K+. The K+ conductance was approximately proportional to the square root of the extracellular K+ concentration. 2 I K(IR) was blocked in a voltage‐dependent manner by external Ba2+ and Cs+, slightly attenuated by 5 mM 4‐aminopyridine (15% inhibition) and insensitive to 10 mM tetra‐ethylammonium, 2 mM Ca2+, 1 mM Cd2+ and 50 μM La3+. 3 In physiological saline, 100 μM Ba2+, which inhibits 86% of IK(IR) at the cell resting potential, depolarized cells by 6.1 ± 0.7 mV from a mean resting potential of −59.6 ± 0.8 mV. 4 Corticotropin releasing hormone (CRH), which activates adenylyl cyclase and stimulates adrenocorticotropic hormone (ACTH) secretion from corticotropes, inhibited IK(IR) by 25% and depolarized the cells by 10.2 ± 1.0 mV. Dibutyryl cAMP ((Bu)2cAMP) mimicked these effects. 5 The membrane depolarization evoked by Ba2+ or CRH increased the cell firing frequency. Comparison of cells exhibiting a membrane potential of approximately −50 mV revealed that spike frequency in the presence of CRH (109 ± 7 spikes (5 min)−1) was greater than in control (60 ± 5 spikes (5 min)−1) or Ba2+‐treated (77 ± 15 spikes (5 min)−1) corticotropes. 6 The data suggest that IK(IR) contributes to maintenance of the resting membrane potential of rat corticotropes. Inhibition of IK(IR) plays a role in, but does not account for all of, the membrane depolarization and enhancement of firing frequency evoked by CRH.

Thyrotropin‐releasing hormone stimulates a calcium‐activated potassium current in a rat anterior pituitary cell line.

1. The ‘giga‐seal’ patch‐electrode technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was used for constant current and voltage‐clamp recordings in the GH3 rat anterior pituitary cell line. 2. Thyrotropin‐releasing hormone (TRH) causes a membrane hyperpolarization that is mediated by a selective increase in K+ permeability. The hyperpolarization cannot be evoked when the cell is internally perfused with a Ca2+ chelator but persists when the external solution that bathes the cell is Ca2+‐free or contains a Ca2+‐channel blocker. 3. Under voltage clamp the TRH‐induced current is approximately linear at negative potentials (‐90 to ‐30 mV) but markedly enhanced at voltages above ‐30 mV). Thus, the affected conductance has a voltage‐dependent component. 4. The TRH‐induced increase in K+ permeability is sensitive to inhibition by 30 mM‐TEA and 200 nM‐apamin, inhibitors of two distinct Ca2+‐activated K+ permeabilities in GH3 cells. 5. The time course of the TRH‐induced K+ current is similar to the time course of a TRH‐induced transient peak elevation of cytosolic Ca2+ that is due to mobilization of Ca2+ from intracellular stores. 6. The effects of TRH on the K+ current and the rise in cytosolic Ca2+ are half‐maximal at 7 nM and 1.7 nM, respectively. 7. It is concluded that the TRH‐induced hyperpolarization is mediated by two distinct Ca2+‐activated K+ conductances that are activated by release of Ca2+ from an intracellular site.

Corticotropin-releasing hormone and calcium signaling in corticotropes

Corticotropin-releasing hormone (CRH) stimulates ACTH secretion from anterior pituitary corticotropes, largely, but possibly not exclusively, via activation of the adenylyl cyclase cascade. CRH stimulates secretion by increasing Ca(2+) influx and by Ca(2+)-independent mechanisms. As Ca(2+) influx is largely regulated by membrane electrical properties, we review the effects of CRH on membrane excitability and changes in cytosolic Ca(2+). We also speculate on possible pathways for CRH modulation of exocytosis by Ca(2+) independent mechanisms.

Tetraethylammonium ion sensitivity of a 35-pS CA2(+)-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone.

Single Ca2+-activated K+ channels were studied in membrane patches from the GH3 anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K+, respectively) Ca2+-activated K+ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca2+-activated K+ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca2+ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca2+ is low; at positive membrane potentials, this channel requires Ca2+ concentrations greater than 10 μM for activation. However, 35-pS channels have a much higher sensitivity to Ca2+ in the first minute after excision (activated by 1 μM Ca2+ at −50 mV). Therefore, it is possible that the Ca2+ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca2+ concentration and by Ca2+ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (Kd=17 mM) and is relatively resistant to inhibition by apamin.

Multiple conductance levels of the dihydropyridine-sensitive calcium channel in GH3 cells

SummaryCalcium channels in GH3 cells exhibit at least five conductance levels when examined in cell-attached or outside-out patches. These channels resemble the high threshold Ca2+ current in their range of activation and inactivation, and in their sensitivity to dihydropyridines (DHP). Mean open times for the five levels were brief (<1 msec) in control solutions but increased in the presence of BAY K 8644. In 100mm Ba2+ and BAY K 8644, the five predominant slope conductances were 8–9, 12–13, 16–18, 23–24, and 28 pS. The present study is the first report of multiple levels of the DHP-sensitive Ca2+ channel occurring with high frequency in native membranes. The range of conductance levels that we observed encompasses the range of conductances found for two other different types of Ca2+ channels and indicates that unit conductance should be used with caution as a distinguishing characteristic for identification of different channel types.

P2X7 Receptor Stimulation of Membrane Internalization in a Thyrocyte Cell Line

Using fluorescent membrane markers, we have previously shown that extracellular ATP stimulates both exocytosis and membrane internalization in the Fisher rat thyroid cell line FRTL. In this study, we examine the actions of ATP using whole-cell recording conditions that favor stimulation of membrane internalization. ATP stimulation of the P2X7 receptor activated a reversible, Ca2+-permeable, cation conductance that slowly increased in size without changes in ion selectivity. ATP also induced a delayed irreversible decrease in cell capacitance (Cm) that was equivalent to an 8% decrease in membrane surface area. Addition of guanosine 5′-0-2-thiodiphosphate to the pipette solution inhibited the ATP-induced decrease in Cm without affecting channel activation. The effects of ATP on membrane conductance were mimicked by 2′,3′-O-(4-benzoylbenzoyl)-ATP, but not by UTP, adenosine, or 2-methylthio-ATP, and were inhibited by pyridoxal phosphate-6-azophenyl-2′4′-disulfonic acid, adenosine 5′-triphosphate-2′3′-dialdehyde, and Cu2+. The capacitance decrease persisted in Na+-, Ca2+- and Cl−-free external saline or with Ca2+-free pipette solution. It is concluded that ATP activation of the inotropic P2X7 receptor stimulates membrane internalization by a mechanism that involves intracellular GTP, but does not require internal Ca2+ or influx of Na+ or Ca2+ through the receptor-gated channel.

Expression of the L-Type Ca2+ Channel in AtT-20 Cells Is Regulated by Cyclic AMP

Activation of adenylyl cyclase by corticotropin-releasing hormone (CRH) stimulates secretion of adrenocorticotropin (ACTH) in rat anterior pituitary corticotropes and in the murine AtT-20 cell line. The stimulation of secretion is mediated by cAMP and is largely dependent on Ca²⁺ influx through voltage-gated L-type Ca²⁺ channels. To investigate whether CRH and cAMP also increase expression of the L-type Ca²⁺ channel in AtT-20 cells, an RNase protection assay was used to measure the α_1C mRNA that encodes the pore-forming subunit of the L-type Ca²⁺ channel. The α_1C mRNA level was measured by autoradiographic densitometry and normalized to the β-actin mRNA level in the same sample. The α_1C mRNA was not changed by 24-hour treatment with CRH (10–500 nM). A 24-hour treatment with 1 mM 8Br-cAMP significantly increased the α_1C mRNA by 40% over its control. The stimulatory effect was blocked by 2 µM actinomycin D and was, therefore, dependent on gene transcription. The measured half-life of the α_1C mRNA, after inhibition of transcription, was 4.7 ± 0.3 h in control and 5.2 ± 0.6 h in the presence of 8Br-cAMP. Thus the 8Br-cAMP- induced increase in α_1C mRNA could be due to an increase in α_1C gene transcription or to a transcriptionally regulated increase in a protein that helps to stabilize α_1C mRNA. Finally, to determine if the increased mRNA was followed by an increase in production of L-type Ca²⁺ channels, the binding of [³H]PN200-110 to Ca²⁺ channel proteins was assayed in AtT-20 membrane fragments. 8Br-cAMP increased [³H]PN200-110 binding sites by 32% (B_max 36.0 ± 1.2 fmol/mg protein in control vs. 47.4 ± 3.2 fmol/mg protein in 8Br-cAMP-treated cells) but did not change the K_d. These studies show that both α_1C mRNA and L-type Ca²⁺ channel protein are increased in AtT-20 cells by cAMP.