Sex differences in pituitary corticotroph excitability.

Stress-related illness represents a major burden on health and society. Sex differences in stress-related disorders are well documented, with women having twice the lifetime rate of depression compared to men and most anxiety disorders. Anterior pituitary corticotrophs are central components of the hypothalamic-pituitary-adrenal (HPA) axis, receiving input from hypothalamic neuropeptides corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), while regulating glucocorticoid output from the adrenal cortex. The dynamic control of electrical excitability by CRH/AVP and glucocorticoids is critical for corticotroph function; however, whether corticotrophs contribute to sexually differential responses of the HPA axis, which might underlie differences in stress-related disorders, is very poorly understood. Using perforated patch clamp electrophysiology in corticotrophs from mice expressing green fluorescent protein under the control of the Pomc promoter, we characterized basal and secretagogue-evoked excitability. Both male and female corticotrophs show predominantly single-spike action potentials under basal conditions; however, males predominantly display spikes with small-amplitude (<20 mV) afterhyperpolarizations (B-type), whereas females displayed a mixture of B-type spikes and spikes with a large-amplitude (>25 mV) afterhyperpolarization (A-type). In response to CRH, or CRH/AVP, male cells almost exclusively transition to a predominantly pseudo-plateau bursting, whereas only female B-type cells display bursting in response to CRH±AVP. Treatment of male or female corticotrophs with 1 nM estradiol (E2) for 24-72 h has no effect on the proportion of cells with A- or B-type spikes in either sex. However, E2 results in the cessation of CRH-induced bursting in both male and female corticotrophs, which can be partially reversed by adding a BK current using a dynamic clamp. RNA-seq analysis of purified corticotrophs reveals extensive differential gene expression at the transcriptional level, including more than 71 mRNAs encoding ion channel subunits. Interestingly, there is a two-fold lower level (p < 0.01) of BK channel pore-forming subunit (Kcnma1) expression in females compared to males, which may partially explain the decrease in CRH-induced bursting. This study identified sex differences at the level of the anterior pituitary corticotroph ion channel landscape and control of both spontaneous and CRH-evoked excitability. Determining the mechanisms of sex differences of corticotroph and HPA activity at the cellular level could be an important step for better understanding, diagnosing, and treating stress-related disorders.

Oxidation modulates LINGO2-induced inactivation of large conductance, Ca2+-activated potassium channels.

Ca2+ and voltage-activated K+ (BK) channels are ubiquitous ion channels that can be modulated by accessory proteins, including ß, γ, and LINGO1 BK subunits. In this study, we utilized a combination of site-directed mutagenesis, patch clamp electrophysiology, and molecular modeling to investigate if the biophysical properties of BK currents were affected by coexpression of LINGO2 and to examine how they are regulated by oxidation. We demonstrate that LINGO2 is a regulator of BK channels, since its coexpression with BK channels yields rapid inactivating currents, the activation of which is shifted ∼-30 mV compared to that of BKα currents. Furthermore, we show the oxidation of BK:LINGO2 currents (by exposure to epifluorescence illumination or chloramine-T) abolished inactivation. The effect of illumination depended on the presence of GFP, suggesting that it released free radicals which oxidized cysteine or methionine residues. In addition, the oxidation effects were resistant to treatment with the cysteine-specific reducing agent DTT, suggesting that methionine rather than cysteine residues may be involved. Our data with synthetic LINGO2 tail peptides further demonstrate that the rate of inactivation was slowed when residues M603 or M605 were oxidized, and practically abolished when both were oxidized. Taken together, these data demonstrate that both methionine residues in the LINGO2 tail mediate the effect of oxidation on BK:LINGO2 channels. Our molecular modeling suggests that methionine oxidation reduces the lipophilicity of the tail, thus preventing it from occluding the pore of the BK channel.

Corticotroph isolation from Pomc-eGFP mice reveals sustained transcriptional dysregulation characterising a mouse model of glucocorticoid-induced suppression of the hypothalamus-pituitary-adrenal axis.

Glucocorticoids (GC) are prescribed for periods > 3 months to 1%-3% of the UK population; 10%-50% of these patients develop hypothalamus-pituitary-adrenal (HPA) axis suppression, which may last over 6 months and is associated with morbidity and mortality. Recovery of the pituitary and hypothalamus is necessary for recovery of adrenal function. We developed a mouse model of dexamethasone (DEX)-induced HPA axis dysfunction aiming to further explore recovery in the pituitary. Adult male wild-type C57BL6/J or Pomc-eGFP transgenic mice were randomly assigned to receive DEX (approximately 0.4 mg kg-1 bodyweight day-1 ) or vehicle via drinking water for 4 weeks following which treatment was withdrawn and tissues were harvested after another 0, 1, and 4 weeks. Corticotrophs were isolated from Pomc-eGFP pituitaries using fluorescence-activated cell sorting, and RNA extracted for RNA-sequencing. DEX treatment suppressed corticosterone production, which remained partially suppressed at least 1 week following DEX withdrawal. In the adrenal, Hsd3b2, Cyp11a1, and Mc2r mRNA levels were significantly reduced at time 0, with Mc2r and Cyp11a1 remaining reduced 1 week following DEX withdrawal. The corticotroph transcriptome was modified by DEX treatment, with some differences between groups persisting 4 weeks following withdrawal. No genes supressed by DEX exhibited ongoing attenuation 1 and 4 weeks following withdrawal, whereas only two genes were upregulated and remained so following withdrawal. A pattern of rebound at 1 and 4 weeks was observed in 14 genes that increased following suppression, and in six genes that were reduced by DEX and then increased. Chronic GC treatment may induce persistent changes in the pituitary that may influence future response to GC treatment or stress.

LKB1 is the gatekeeper of carotid body chemosensing and the hypoxic ventilatory response.

The hypoxic ventilatory response (HVR) is critical to breathing and thus oxygen supply to the body and is primarily mediated by the carotid bodies. Here we reveal that carotid body afferent discharge during hypoxia and hypercapnia is determined by the expression of Liver Kinase B1 (LKB1), the principal kinase that activates the AMP-activated protein kinase (AMPK) during metabolic stresses. Conversely, conditional deletion in catecholaminergic cells of AMPK had no effect on carotid body responses to hypoxia or hypercapnia. By contrast, the HVR was attenuated by LKB1 and AMPK deletion. However, in LKB1 knockouts hypoxia evoked hypoventilation, apnoea and Cheyne-Stokes-like breathing, while only hypoventilation and apnoea were observed after AMPK deletion. We therefore identify LKB1 as an essential regulator of carotid body chemosensing and uncover a divergence in dependency on LKB1 and AMPK between the carotid body on one hand and the HVR on the other.

Chronic stress facilitates bursting electrical activity in pituitary corticotrophs.

Coordination of an appropriate stress response is dependent upon anterior pituitary corticotroph excitability in response to hypothalamic secretagogues and glucocorticoid negative feedback. A key determinant of corticotroph excitability is large conductance calcium- and voltage-activated (BK) potassium channels that are critical for promoting corticotrophin-releasing hormone (CRH)-induced bursting that enhances adrenocorticotrophic hormone secretion. Previous studies revealed hypothalamic-pituitary-adrenal axis hyperexcitability following chronic stress (CS) is partly a function of increased corticotroph output. Thus, we hypothesise that chronic stress promotes corticotroph excitability through a BK-dependent mechanism. Corticotrophs from CS mice displayed significant increase in spontaneous bursting, which was suppressed by the BK blocker paxilline. Mathematical modelling reveals that the time constant of BK channel activation, plus properties and proportion of BK channels functionally coupled to L-type Ca2+ channels determines bursting activity. Surprisingly, CS corticotrophs (but not unstressed) display CRH-induced bursting even when the majority of BK channels are inhibited by paxilline, which modelling suggests is a consequence of the stochastic behaviour of a small number of BK channels coupled to L-type Ca2+ channels. Our data reveal that changes in the stochastic behaviour of a small number of BK channels can finely tune corticotroph excitability through stress-induced changes in BK channel properties. Importantly, regulation of BK channel function is highly context dependent allowing dynamic control of corticotroph excitability over a large range of time domains and physiological challenges in health and disease. This is likely to occur in other BK-expressing endocrine cells, with important implications for the physiological processes they regulate and the potential for therapy. KEY POINTS: Chronic stress (CS) is predicted to modify the electrical excitability of anterior pituitary corticotrophs. Electrophysiological recordings from isolated corticotrophs from CS male mice display spontaneous electrical bursting behaviour compared to the tonic spiking behaviour of unstressed corticotrophs. The increased spontaneous bursting from CS corticotrophs is BK-dependent and mathematical modelling reveals that the time constant of activation, properties and proportion of BK channels functionally coupled to L-type calcium channels determines the promotion of bursting activity. CS (but not unstressed) corticotrophs display corticotrophin-releasing hormone-induced bursting even when the majority of BK channels are pharmacologically inhibited, which can be explained by the stochastic behaviour of a small number of BK channels with distinct properties. Corticotroph excitability can be finely tuned by the stochastic behaviour of a small number of BK channels dependent on their properties and functional co-localisation with L-type calcium channels to control corticotroph excitability over diverse time domains and physiological challenges.

Glucocorticoid action in the anterior pituitary gland: Insights from corticotroph physiology.

The anterior pituitary is exposed to ultradian, circadian and stress-induced rhythms of circulating glucocorticoid hormones. Glucocorticoids feedback at the level of the pituitary corticotroph to control their own production through multiple mechanisms. This review highlights key insights from analysis of the dynamics of rapid and early glucocorticoid feedback that reveal both non-genomic and genomic mechanisms mediated by glucocorticoid receptors. Importantly, a common target is control of electrical excitability and calcium signalling although non-genomic effects may also involve control of hormone secretion distal to calcium signalling. Understanding the mechanisms and functional consequences of pulsatile glucocorticoid signalling in the anterior pituitary promises to elucidate the role of glucocorticoids in health and disease, as well as identifying potential diagnostic and therapeutic targets.

Regulatory effects of protein S-acylation on insulin secretion and insulin action.

Post-translational modifications (PTMs) such as phosphorylation and ubiquitination are well-studied events with a recognized importance in all aspects of cellular function. By contrast, protein S-acylation, although a widespread PTM with important functions in most physiological systems, has received far less attention. Perturbations in S-acylation are linked to various disorders, including intellectual disability, cancer and diabetes, suggesting that this less-studied modification is likely to be of considerable biological importance. As an exemplar, in this review, we focus on the newly emerging links between S-acylation and the hormone insulin. Specifically, we examine how S-acylation regulates key components of the insulin secretion and insulin response pathways. The proteins discussed highlight the diverse array of proteins that are modified by S-acylation, including channels, transporters, receptors and trafficking proteins and also illustrate the diverse effects that S-acylation has on these proteins, from membrane binding and micro-localization to regulation of protein sorting and protein interactions.

Site-specific deacylation by ABHD17a controls BK channel splice variant activity.

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/ß-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0-S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

LINGO1 is a regulatory subunit of large conductance, Ca2+-activated potassium channels.

LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson's disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca2+-activated K+ (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a "functional knockdown" of these currents and may contribute to the tremor associated with increased LINGO1 levels.

S-Acylation controls functional coupling of BK channel pore-forming α-subunits and ß1-subunits.

The properties and physiological function of pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are potently modified by their functional coupling with regulatory subunits in many tissues. However, mechanisms that might control functional coupling are very poorly understood. Here we show that S-acylation, a dynamic post-translational lipid modification of proteins, of the intracellular S0-S1 loop of the BK channel pore-forming α-subunit controls functional coupling to regulatory ß1-subunits. In HEK293 cells, α-subunits that cannot be S-acylated show attenuated cell surface expression, but expression was restored by co-expression with the ß1-subunit. However, we also found that nonacylation of the S0-S1 loop reduces functional coupling between α- and ß1-subunits by attenuating the ß1-subunit-induced left shift in the voltage for half-maximal activation. In mouse vascular smooth muscle cells expressing both α- and ß1-subunits, BK channel α-subunits were endogenously S-acylated. We further noted that S-acylation is significantly reduced in mice with a genetic deletion of the palmitoyl acyltransferase (Zdhhc23) that controls S-acylation of the S0-S1 loop. Genetic deletion of Zdhhc23 or broad-spectrum pharmacological inhibition of S-acylation attenuated endogenous BK channel currents independently of changes in cell surface expression of the α-subunit. We conclude that functional effects of S-acylation on BK channels depend on the presence of ß1-subunits. In the absence of ß1-subunits, S-acylation promotes cell surface expression, whereas in its presence, S-acylation controls functional coupling. S-Acylation thus provides a mechanism that dynamically regulates the functional coupling with ß1-subunits, enabling an additional level of conditional, cell-specific control of ion-channel physiology.

siRNA Knockdown of Mammalian zDHHCs and Validation of mRNA Expression by RT-qPCR.

The lack of specific pharmacological tools to interrogate the functional role of palmitoyl acyltransferases (zDHHCs) in mammalian cells has significantly hampered the understanding of this important gene family. Gene silencing by RNA interference (RNAi) is a process in eukaryotes that allows specific knockdown of the expression of proteins by targeting their coding mRNA. RNAi can thus be used as a proteomic tool to study the functional role of specific zDHHCs in cells by analyzing the effects of endogenous zDHHC knockdown on their protein targets or pathways. Here we describe the application of short interfering RNA (siRNA), a class of short (20-25 base pairs) double-stranded RNAs, to knockdown endogenous zDHHC enzymes expressed in human embryonic kidney (HEK293) cells and subsequent validation of knockdown efficiency using RT-qPCR to quantify zDHHC mRNA levels.

Control of anterior pituitary cell excitability by calcium-activated potassium channels.

In anterior pituitary endocrine cells, large (BK), small (SK) and intermediate (IK) conductance calcium activated potassium channels are key determinants in shaping cellular excitability in a cell type- and context-specific manner. Indeed, these channels are targeted by multiple signaling pathways that stimulate or inhibit cellular excitability. BK channels can, paradoxically, both promote electrical bursting as well as terminate bursting and spiking dependent upon intrinsic BK channel properties and proximity to voltage gated calcium channels in somatotrophs, lactotrophs and corticotrophs. In contrast, SK channels are predominantly activated by calcium released from intracellular IP3-sensitive calcium stores and mediate membrane hyperpolarization in cells including gonadotrophs and corticotrophs. IK channels are predominantly expressed in corticotrophs where they limit membrane excitability. A major challenge for the future is to determine the cell-type specific molecular composition of calcium-activated potassium channels and how they control anterior pituitary hormone secretion as well as other calcium-dependent processes.

Distinct domains of the ß1-subunit cytosolic N terminus control surface expression and functional properties of large-conductance calcium-activated potassium (BK) channels.

The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory ß1-subunits. Although the short cytosolic N-terminal domain of the ß1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the ß1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the ß1-subunit N terminus.

Heterogeneity of Calcium Responses to Secretagogues in Corticotrophs From Male Rats.

Heterogeneity in homotypic cellular responses is an important feature of many biological systems, and it has been shown to be prominent in most anterior pituitary hormonal cell types. In this study, we analyze heterogeneity in the responses to hypothalamic secretagogues in the corticotroph cell population of adult male rats. Using the genetically encoded calcium indicator GCaMP6s, we determined the intracellular calcium responses of these cells to corticotropin-releasing hormone and arginine-vasopressin. Our experiments revealed marked population heterogeneity in the response to these peptides, in terms of amplitude and dynamics of the responses, as well as the sensitivity to different concentrations and duration of stimuli. However, repeated stimuli to the same cell produced remarkably consistent responses, indicating that these are deterministic on a cell-by-cell level. We also describe similar heterogeneity in the sensitivity of cells to inhibition by corticosterone. In summary, our results highlight a large degree of heterogeneity in the cellular mechanisms that govern corticotroph responses to their physiological stimuli; this could provide a mechanism to extend the dynamic range of the responses at the population level to allow adaptation to different physiological challenges.

Obesogenic and Diabetogenic Effects of High-Calorie Nutrition Require Adipocyte BK Channels.

Elevated adipose tissue expression of the Ca2+- and voltage-activated K+ (BK) channel was identified in morbidly obese men carrying a BK gene variant, supporting the hypothesis that K+ channels affect the metabolic responses of fat cells to nutrients. To establish the role of endogenous BKs in fat cell maturation, storage of excess dietary fat, and body weight (BW) gain, we studied a gene-targeted mouse model with global ablation of the BK channel (BKL1/L1) and adipocyte-specific BK-deficient (adipoqBKL1/L2) mice. Global BK deficiency afforded protection from BW gain and excessive fat accumulation induced by a high-fat diet (HFD). Expansion of white adipose tissue-derived epididymal BKL1/L1 preadipocytes and their differentiation to lipid-filled mature adipocytes in vitro, however, were improved. Moreover, BW gain and total fat masses of usually superobese ob/ob mice were significantly attenuated in the absence of BK, together supporting a central or peripheral role for BKs in the regulatory system that controls adipose tissue and weight. Accordingly, HFD-fed adipoqBKL1/L2 mutant mice presented with a reduced total BW and overall body fat mass, smaller adipocytes, and reduced leptin levels. Protection from pathological weight gain in the absence of adipocyte BKs was beneficial for glucose handling and related to an increase in body core temperature as a result of higher levels of uncoupling protein 1 and a low abundance of the proinflammatory interleukin-6, a common risk factor for diabetes and metabolic abnormalities. This suggests that adipocyte BK activity is at least partially responsible for excessive BW gain under high-calorie conditions, suggesting that BK channels are promising drug targets for pharmacotherapy of metabolic disorders and obesity.

Glucocorticoids Inhibit CRH/AVP-Evoked Bursting Activity of Male Murine Anterior Pituitary Corticotrophs.

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which governs the neuroendocrine response to stress. Corticotrophs are electrically excitable and fire spontaneous single-spike action potentials and also display secretagogue-induced bursting behavior. The HPA axis function is dependent on effective negative feedback in which elevated plasma glucocorticoids result in inhibition at the level of both the pituitary and the hypothalamus. In this study, we have used an electrophysiological approach coupled with mathematical modeling to investigate the regulation of spontaneous and CRH/arginine vasopressin-induced activity of corticotrophs by glucocorticoids. We reveal that pretreatment of corticotrophs with 100 nM corticosterone (CORT; 90 and 150 min) reduces spontaneous activity and prevents a transition from spiking to bursting after CRH/arginine vasopressin stimulation. In addition, previous studies have identified a role for large-conductance calcium- and voltage-activated potassium (BK) channels in the generation of secretagogue-induced bursting in corticotrophs. Using the dynamic clamp technique, we demonstrated that CRH-induced bursting can be switched to spiking by subtracting a fast BK current, whereas the addition of a fast BK current can induce bursting in CORT-treated cells. In addition, recordings from BK knockout mice (BK(-/-)) revealed that CORT can also inhibit excitability through BK-independent mechanisms to control spike frequency. Thus, we have established that glucocorticoids can modulate multiple properties of corticotroph electrical excitability through both BK-dependent and BK-independent mechanisms.

The physiology of protein S-acylation.

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field.

Large conductance Ca²âº-activated K⁺ (BK) channels promote secretagogue-induced transition from spiking to bursting in murine anterior pituitary corticotrophs.

Anterior pituitary corticotroph cells are a central component of the hypothalamic-pituitary-adrenal (HPA) axis essential for the neuroendocrine response to stress. Corticotrophs are excitable cells that receive input from two hypothalamic secretagogues, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) to control the release of adrenocorticotrophic hormone (ACTH). Although corticotrophs are spontaneously active and increase in excitability in response to CRH and AVP the patterns of electrical excitability and underlying ionic conductances are poorly understood. In this study, we have used electrophysiological, pharmacological and genetic approaches coupled with mathematical modelling to investigate whether CRH and AVP promote distinct patterns of electrical excitability and to interrogate the role of large conductance calcium- and voltage-activated potassium (BK) channels in spontaneous and secretagogue-induced activity. We reveal that BK channels do not play a significant role in the generation of spontaneous activity but are critical for the transition to bursting in response to CRH. In contrast, AVP promotes an increase in single spike frequency, a mechanism independent of BK channels but dependent on background non-selective conductances. Co-stimulation with CRH and AVP results in complex patterns of excitability including increases in both single spike frequency and bursting. The ability of corticotroph excitability to be differentially regulated by hypothalamic secretagogues provides a mechanism for differential control of corticotroph excitability in response to different stressors.

Large conductance Ca2+ -activated K+ channels (BK) promote secretagogue-induced transition from spiking to bursting in murine anterior pituitary corticotrophs.

Anterior pituitary corticotroph cells are a central component of the hypothalamic-pituitary-adrenal (HPA) axis essential for the neuroendocrine response to stress. Corticotrophs are excitable cells that receive input from two hypothalamic secretagogues, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) to control the release of adrenocorticotrophin hormone (ACTH). Although corticotrophs are spontaneously active and increase in excitability in response to CRH and AVP the patterns of electrical excitability and underlying ionic conductances are poorly understood. In this study, we have used electrophysiological, pharmacological and genetic approaches coupled with mathematical modeling to investigate whether CRH and AVP promote distinct patterns of electrical excitability and to interrogate the role of large conductance calcium- and voltage-activated (BK) channels in spontaneous and secretagogue-induced activity. We reveal that BK channels do not play a significant role in the generation of spontaneous activity but are critical for the transition to bursting in response to CRH. In contrast, AVP promotes an increase in single spike frequency, a mechanism independent of BK channels but dependent on background non-selective conductances. Co-stimulation with CRH and AVP results in complex patterns of excitability including increases in both single spike frequency and bursting. The ability of corticotroph excitability to be differentially regulated by hypothalamic secretagogues provides a mechanism for differential control of corticotroph excitability in response to different stressors. This article is protected by copyright. All rights reserved.