Non-technical summaryOur ability to respond to stress is critically dependent upon the release of the stress hormone adrenocorticotrophic hormone (ACTH) from corticotroph cells of the anterior pituitary gland. ACTH release is controlled by the electrical properties of corticotrophs that are determined by the movement of ions through channel pores in the plasma membrane. We show that a calcium-activated potassium ion channel called SK4 is expressed in corticotrophs and regulates ACTH release. We provide evidence of how SK4 channels control corticotroph function, which is essential for understanding homeostasis and for treating stress-related disorders.AbstractThe anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic–pituitary–adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4−/−). In addition, Kcnn4−/− mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4−/− mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.

Membrane-associated receptors for rapid, steroidal neuromodulation remain elusive. Estradiol has been reported to facilitate activation of voltage- and Ca(2+)-dependent BK potassium channels encoded by Slo, if associated with beta1 subunits. We show here that 1) multiple members of the beta family confer sensitivity to multiple steroids on BK channels, 2) that beta subunits differentiate between steroids, and 3) that different betas have distinct relative preferences for particular steroids. Expressed in HEK 293 cells, inside-out patches with channels composed of Slo-alpha alone showed no steroid sensitivity. Cells expressing alphabeta4 exhibited potent, rapid, reversible, and dose-dependent potentiation by corticosterone (CORT; a glucocorticoid), and were potentiated to a lesser degree by other sex and stress steroids. In contrast, alphabeta2 channels were potentiated more strongly by dehydroepiandrosterone (DHEA; an enigmatic, stress-related adrenal androgen), and to a lesser extent by CORT, estradiol, testosterone, and DHEA-S. Cholesterol had no effect on any BK channel compositions tested. Conductance-voltage plots of channels composed of alpha plus beta2 or beta4 subunits were shifted in the negative direction by steroids, indicating greater activation at negative voltages. Thus our results argue that the variety of Slo-beta subunit coexpression patterns occurring in vivo expands the repertoire of Slo channel gating in yet another dimension not fully appreciated, rendering BK gating responsive to dynamic fluctuations in a multiple of steroid hormones.

Although adrenal glucocorticoids cortisol and corticosterone (CORT) have numerous "genomic" effects on adrenomedullary chromaffin cells, acute modulatory actions remain largely unknown, despite rapid stress-related changes in secretion. We report that 1 microM glucocorticoids rapidly modulate gating of chromaffin cell BK channels and action potential firing. In general, CORT, or the analog dexamethasone (DEX), increased channel activity in inside-out bovine patches, an effect not blocked by the glucocorticoid receptor (GR) antagonist RU38486. By contrast, these steroids could profoundly inhibit BK activation in many rat patches, while facilitating activation in others. We show that BK inhibition arises from a negative shift in the voltage dependence of BK inactivation paralleling that for activation. We report that rat cells characteristically exhibit greater repetitive firing ability than bovine cells in the absence of glucocorticoids. In both species, steroid application typically increased firing responses to smaller current injections, attributable to BK-enhanced repolarization and Na+ channel deinactivation. However, in rat cells, where BK inactivation is generally faster and more complete, glucocorticoids tended to dampen responses to stronger stimuli. Thus, in the context of natural variation in BK gating, glucocorticoids can either promote or limit firing responses. We suggest that steroids exploit BK gating variety to tailor catecholamine output in a species- and context-specific fashion.

Large conductance calcium-activated potassium (BK) channels are very prominently expressed in adrenal chromaffin and many anterior pituitary cells, where they shape intrinsic excitability complexly. Stress- and sex-steroids regulate alternative splicing of Slo-alpha, the pore-forming subunit of BK channels, and chronic behavioural stress has been shown to alter Slo splicing in tree shrew adrenals. In the present study, we focus on mice, measuring the effects of chronic behavioural stress on total mRNA expression of the Slo-alpha gene, two key BK channel beta subunit genes (beta2 and beta4), and the 'STREX' splice variant of Slo-alpha. As a chronic stressor, males of the relatively aggressive SJL strain were housed with a different unfamiliar SJL male every 24 h for 19 days. This 'social-instability' paradigm stressed all individuals, as demonstrated by reduced weight gain and elevated corticosterone levels. Five quantitative reverse transcriptase-polymerase chain assays were performed in parallel, including beta-actin, each calibrated against a dilution series of its corresponding cDNA template. Stress-related changes in BK expression were larger in mice tested at 6 weeks than 9 weeks. In younger animals, Slo-alpha mRNA levels were elevated 44% and 116% in the adrenal medulla and pituitary, respectively, compared to individually-housed controls. beta2 and beta4 mRNAs were elevated 162% and 194% in the pituitary, but slightly reduced in the adrenals of stressed animals. In the pituitary, dominance scores of stressed animals correlated negatively with alpha and beta subunit expression, with more subordinate individuals exhibiting levels that were three- to four-fold higher than controls or dominant individuals. STREX variant representation was lower in the subordinate subset. Thus, the combination of subunits responding to stress differs markedly between adrenal and pituitary glands. These data suggest that early stress will differentially affect neuroendocrine cell excitability, and call for detailed analysis of functional consequences.

B/W photographs. 31-item bibliography, discography and web site listing. Region: New Hampshire. Genres: contra dances and square dances. 16 tracks recorded in the studio. 67:00.1999. Choose Your Partners!, produced in collaboration with the New Hampshire Council of the Arts, documents a tradition of contra-and square-dance music which has been particularly well preserved in dance halls of the southern Monadnock region of New Hampshire. The recording quality is rich and clear, and the liner notes are extensive and informative, including a detailed bibliography of books, recordings, performers and websites. The included essay (by Jack Beard, Lynn Martin, and Kate Van Winkle Kellar) emphasizes the participatory aspect of the music, with references to the"spring of an old wooden floor," and indications of metrical shifts which would enliven actual dancers. Synchronic differences, such as regional variations, typically emphasized in collections of this sort, are given less prominence than historical variation. Rather than posit an unchanged "found artifact," the collection documents stylistic changes over the past 150 years: for example, the effects of the vacationing skiing community, which reportedly resulted in increased complexity of melodies and harmonies, and larger repertoires. Contemporary compositions (by such well-known dance musicians as Bob McQuillen) and complex medleys are also included as the latest link in this evolving tradition. Notes for each selection feature the tune name(s), dance type and a short paragraph describing the general nature of the dances themselves. The inclusion of dance callers announcing the steps helps make up for the relative lack of visual aids, as does the occasional sound of a clogger's steps, or occasional "spontaneous" applause at the end of a medley.