Control of alternative splicing of potassium channels by stress hormones.

Many molecular mechanisms for neural adaptation to stress remain unknown. Expression of alternative splice variants of Slo, a gene encoding calcium- and voltage-activated potassium channels, was measured in rat adrenal chromaffin tissue from normal and hypophysectomized animals. Hypophysectomy triggered an abrupt decrease in the proportion of Slo transcripts containing a "STREX" exon. The decrease was prevented by adrenocorticotropic hormone injections. In Xenopus oocytes, STREX variants produced channels with functional properties associated with enhanced repetitive firing. Thus, the hormonal stress axis is likely to control the excitable properties of epinephrine-secreting cells by regulating alternative splicing of Slo messenger RNA.

Serotonin selectively inhibits growth cone motility and synaptogenesis of specific identified neurons.

The motile activity of growth cones of specific identified neurons is inhibited by the neurotransmitter serotonin, although other identified neurons are unaffected. As a consequence, affected neurons are unable to form electrical synapses, whereas other neurons whose growth is unaffected can still interconnect. This result demonstrates that neurotransmitters can play a prominent role in regulating neuronal architecture and connectivity in addition to their classical role in neurotransmission.

mSlo, a complex mouse gene encoding "maxi" calcium-activated potassium channels.

Complementary DNAs (cDNAs) from mSlo, a gene encoding calcium-activated potassium channels, were isolated from mouse brain and skeletal muscle, sequenced, and expressed in Xenopus oocytes. The mSlo-encoded channel resembled "maxi" or BK (high conductance) channel types; single channel conductance was 272 picosiemens with symmetrical potassium concentrations. Whole cell and single channel currents were blocked by charybdotoxin, iberiotoxin, and tetraethylammonium ion. A large number of variant mSlo cDNAs were isolated, indicating that several diverse mammalian BK channel types are produced by a single gene.

Action potential waveform voltage-clamp commands reveal striking differences in calcium entry via low and high voltage-activated calcium channels.

Calcium channels transduce natural voltage transients, like action potentials, into functionally important intracellular calcium transients. We have used digitally constructed waveforms that simulate natural action potentials as voltage-clamp commands to study channel function in transduction. Whole-cell calcium currents elicited by several action potential waveforms (APWs) were studied. The currents were subdivided into T (or low voltage-activated) and high voltage-activated components. Calcium entry through T channels constituted a disproportionately large fraction of the total during normal, brief APWs. Entry through high voltage-activated channels was much more responsive to APW, increasing more significantly as APW duration increased. Thus the results indicate that differences in the gating properties of these two channel classes combine to endow them with strikingly different transducer properties.

Development alters the expression of calcium currents in chick limb motoneurons.

Calcium current types expressed in vertebrate spinal motoneurons have not been previously resolved. We have resolved three types in chick limb motoneurons identified by retrograde labeling and report that dramatic changes in their expression take place during development in vivo. T-, N-, and L-type calcium currents were distinguished on the basis of kinetics, voltage dependence, and unique pharmacological sensitivities. Developmental changes were characterized by studying motoneurons isolated from embryos at three stages spanning neuromuscular system development. T currents were dominant at the earliest stage. Motoneurons from embryos 2 days older showed much reduced T currents and much increased N and L currents. We suggest that mature motoneurons will be dominated by N- and L-type calcium currents and that T current may serve developmental roles.

Interactive effects of serotonin and acetylcholine on neurite elongation.

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.

Pituitary control of BK potassium channel function and intrinsic firing properties of adrenal chromaffin cells.

The discovery that the hypothalamic-pituitary-adrenocortical (HPA) endocrine stress axis controls an alternative splicing decision in chromaffin Slo-encoded BK (big potassium) channels raised the possibility that activation of the HPA could serve as a mechanism to tune the intrinsic electrical properties of epinephrine-secreting adrenal chromaffin cells. To test this, we compared BK functional properties and cell excitability in chromaffin cells from normal and hypophysectomized (pituitary-ablated) rats. Hypophysectomy was found to alter the voltage dependence and kinetics of BK gating, making channels less accessible for activation from rest. Perforated-patch recordings revealed changes in action potential waveform and repetitive firing properties. The maximum number of spikes that could be elicited with a 2 sec depolarizing current pulse was reduced by approximately 50% by hypophysectomy. The results indicate that pituitary hormones can adapt the mechanics of adrenal catecholamine release by tailoring BK channel function.

Regulation of Slo potassium channel alternative splicing in the pituitary by gonadal testosterone.

Slo gene-encoded BK potassium channels are prominent in both adrenomedullary and pituitary tissues. At one alternative splicing site, both tissues express variants with and without the optional 'STREX' exon. In adrenal chromaffin cells, this splicing choice, which has important ramifications for cell excitability, has been shown to be regulated by steroid hormones, including glucocorticoids and adrenal androgens. Moreover, striking sex differences are seen between male and female tree shrews. Here, we test the hypothesis that gonadal testosterone regulates splicing in these tissues. No significant sex differences were found in rats, in either adrenals or pituitaries. In the adrenal medulla, prepubertal castration in male rats increased the relative abundance of STREX transcripts slightly, but not significantly, as measured several weeks after puberty. However, castration substantially decreased STREX representation in the rat pituitary. Silastic implants of testosterone inserted at castration prevented this STREX decline. In postpubescent males, castration or T implants had less effect. Thus, we report (i) steroidal regulation of Slo splicing in the pituitary and (ii) participation of the male gonads in this regulation.

Pituitary network connectivity as a mechanism for the luteinising hormone surge.

Ovulation in vertebrates is caused by a surge of luteinising hormone (LH) from the pituitary. The LH surge is initiated by rising oestradiol concentration, although the precise mechanism of oestradiol action in humans and primates is not yet understood. Recent advances in labelling and three-dimensional imaging have revealed a rich pituitary structure of interwoven networks of different cell types. In the present study, we develop a mathematical model to test the hypothesis that oestradiol modulation of connectivity between pituitary cells can underlie the LH surge. In the model, gonadotrophin-releasing hormone (GnRH) pulses stimulate LH secretion by two independent mechanisms. The first mechanism corresponds to the well known direct action of GnRH on gonadotrophs, which is inhibited by the rising oestradiol concentration. The second mechanism of GnRH action is to stimulate a recurrent network of pituitary cells; in this case, the folliculostellate cells, which in turn stimulate LH secretion from the gonadotrophs. The network activity is modelled by a one-dimensional ordinary differential equation. The key to the LH surge in the model lies in the assumption that oestradiol modulates network connectivity. When the circulating oestradiol concentration is low, the network is barely connected, and cannot maintain a recurrent signal. When the oestradiol concentration is high, the network is highly connected, and maintains a high level of activity even after GnRH stimulation, thereby leading to a surge of LH secretion.

Social stress alters expression of large conductance calcium-activated potassium channel subunits in mouse adrenal medulla and pituitary glands.

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.

Beta2 and beta4 subunits of BK channels confer differential sensitivity to acute modulation by steroid hormones.

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.

Membrane voltage and neurotransmitter regulation of neuronal growth cone motility.

The neurotransmitters serotonin and dopamine inhibit growth cone motility and neurite elongation of specific identified neurons of the pond snail Helisoma. Similarly, experimentally evoked action potentials inhibit motility of these growth cones. Here we explore the possibility that the motility- and elongation-inhibiting actions of serotonin and dopamine derive from the electrophysiological responses of the respective neurons. Evidence of three types in support of this hypothesis is presented: (1) Only those identified neurons for which motility is inhibited by serotonin or dopamine respond to the transmitter with sustained electrical excitation. (2) The magnitude of the electrical excitation response correlates with the degree of inhibition of growth cone motility. (3) The injection of hyperpolarizing current enables motility to continue as in the absence of transmitters. We conclude that membrane voltage is an important level of control of growth cone motility, at which neurotransmitters exert a regulatory influence.

The genesis of membrane bone in the embryonic chick maxilla: epithelial-mesenchymal tissue recombination studies.

In the present study, the question of whether a relatively non-specific epithelial requirement exists for membrane bone formation within the maxillary mesenchyme was investigted. Organ rudiments from embryonic chicks of three to five days of incubation (HH 18-25) were enzymatically separated into the epithelial and mesenchymal components. Maxillary mesenchyme (from embryos HH 18-19) which in the absence of epithelium will not form bone was recombined with epithelium from maxillae of similarly aged embryos (homotypic-homochronic recombination) and of older embryos (HH 25) (homotypic-heterochronic recombination). Heterotypic recombinations were made between maxillary mesenchyme (HH 18-19) and the epithelium from wing and hind-limb buds (HH 19-22). Recombinants were grown as grafts on the chorioallantoic membranes of host chick embryos. Grafts of intact macillae, isolated maxillary mesenchyme, and isolated epithelia from the maxilla, wing-, and hind-limb buds were grown as controls. The histodifferentiation of grafted intact maxillae was similar to that in vivo; both cartilage and membrane bone differentiated within the mesenchyme. Grafts of maxillary mesenchyme (from embryos HH 18-19) grown in the absence of epithelium formed cartilage but did not form membrane bone. Grafts of maxillary mesenchyme (from embryos HH 18-19) recombined with epithelium in homotypic-homochronic, homotypic-heterochronic, and heterotypic tissue combinations formed membrane bone in addition to cartilage. These results indicate that maxillary mesenchyme requires the presence of epithelium to promote osteogenesis and that this epithelial requirement is relatively non-specific in terms of type and age of epithelium.

Dopamine and serotonin inhibition of neurite elongation of different identified neurons.

This study demonstrates that a second classical neurotransmitter, dopamine, can act to suppress regenerative neurite outgrowth. Single identified neurons were dissected from two central ganglia of the snail Helisoma, and growth cone motility was studied as neurites regenerated in cell culture. Both dopamine and serotonin inhibited growth cone motility and elongation of neurites. Outgrowth inhibition ranged from sustained arrest to a similar but transient response. The effects of dopamine and serotonin are neuron-selective. Specific neurons affected by dopamine and serotonin represent distinct sets. One neuron was found that responds to both agents. The implications of neurotransmitter regulation of the dynamics of neuronal morphology are discussed.

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin.

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.

Bovine versus rat adrenal chromaffin cells: big differences in BK potassium channel properties.

Both bovine and rat adrenal chromaffin cells have served as pioneering model systems in cellular neurophysiology, including in the study of large conductance calcium- and voltage-dependent K(+) (BK) channels. We now report that while BK channels dominate the outward current profile of both species, specific gating properties vary widely across cell populations, and the distributions of these properties differ dramatically between species. Although BK channels were first described in bovine chromaffin cells, rapidly inactivating ones were discovered in rat chromaffin cells. We report that bovine cells can also exhibit inactivating BK channels with varying properties similar to those in rat cells. However, a much smaller proportion of bovine cells exhibit inactivating BK current, the proportion of the total current that inactivates is usually smaller, and the rate of inactivation is often much slower. Other gating features differ as well; the voltage dependence of channel activation is much more positive for bovine cells, and their rates of activation and deactivation are faster and slower, respectively. Modeling studies suggest that channel heterogeneity is consistent with varying tetrameric combinations of inactivation-competent versus -incompetent subunits. The results suggest that chromaffin BK channel functional nuances represent an important level for evolutionary tailoring of autonomic stress responses.

The differentiation of excitability in embryonic chick limb motoneurons.

The well-documented role of neuromuscular activity as a regulator of motoneuron and muscle development raises important questions about the differentiation of excitability in motoneurons. We have recently described changes in expression of voltage-dependent calcium currents that take place during neuromuscular development in the chick embryo (McCobb et al., 1989). We now report similar analyses, using whole-cell patch-recording methods, of the major currents underlying action potential generation in the same motoneurons. Studies were conducted on identified hindlimb motoneurons isolated from the spinal cord at 3 very different stages of chick hindlimb development. Motoneurons could generate overshooting action potentials at the earliest stage studied [embryonic day 4 (E4)]. However, large changes in densities of several voltage-dependent ionic currents occurred thereafter. E6 and E11 motoneurons had progressively larger INa densities and, consequently, greater action potential amplitudes. Densities of 2 potassium currents, Ik and IA, increased on separate schedules. The relatively late and much larger increase in IA resulted in a substantial developmental decline in action potential duration. These changes, which will greatly affect motoneuron output to muscle by affecting Ca2+ entry through voltage-gated channels, occur at the same time that activity-dependent developmental changes occur in the neuromuscular system.

A human calcium-activated potassium channel gene expressed in vascular smooth muscle.

Large-conductance Ca(2+)-activated K+ (BK) channels are widespread and functionally heterogeneous. In other classes of K+ channels, functional heterogeneity derives from large gene families, alternative splicing, heterologous subunit composition, and functional modulation. The molecular basis of mammalian BK channel heterogeneity is unknown, since only a single gene (mSlo) has been identified. BK channels in native vascular smooth muscle have an apparent Ca2+ sensitivity approximately 10-fold greater than native brain or skeletal muscle channels, or cloned mSlo channels. Using mSlo as a low-stringency probe, we screened human arterial smooth muscle and genomic libraries extensively in search of genes or splice variants with novel properties. We isolated the human homologue of mSlo, including two novel splice variant forms, but found no other related genes. Electrophysiological characterization of the hSlo clones in Xenopus oocytes and Chinese hamster ovary cells gave BK currents that were not measurably different from mSlo currents. However, coexpression of hSlo with a recently cloned beta-subunit derived from smooth muscle dramatically increased apparent Ca2+ sensitivity. Thus alpha-subunits alone may not determine Ca2+ sensitivity of vascular smooth muscle BK channels. hSlo was mapped to human chromosome 10q23.1, and the genomic structure was analyzed. Immediately after the amino terminal, two unusual regions of trinucleotide repeating sequences are present. The first of these regions encodes polyglycine, and the second encodes polyserine. Both regions of repeated sequence are conserved between the mouse and human genome.

Neuron-specific growth cone properties as seen in identified neurons of Helisoma.

By using cell-cultured identified neurons of the snail Helisoma, we demonstrate that the growth cones of different neurons are intrinsically different from one another in terms of their structure, behavior, and response to environmental signals. Structurally neuron 5 has a greater number of filopodia per growth cone, has shorter filopodia, and has a smaller interfilopodial distance than neuron 19. Behaviorally, the growth cones of neuron 5 advance over the substratum at a faster rate than those of neuron 19; and the growth cones of neuron 19, but not of neuron 5, respond to the presence of serotonin in their environment by retracting their filopodia. In addition to such intrinsic differences between the growth cones of different neurons, we also demonstrate that the separate growth cones of a single neuron, while having identical properties, can act independently of one another. Focal application of serotonin to a growth cone causes only a localized retraction of that growth cones' filopodia. Other growth cones that are attached to the same neuron but that are not exposed to serotonin retain their normal structural features.