Molecular moieties masking Ca2+-dependent facilitation of voltage-gated Cav2.2 Ca2+ channels.

Voltage-gated Cav2.1 (P/Q-type) Ca2+ channels undergo Ca2+-dependent inactivation (CDI) and facilitation (CDF), both of which contribute to short-term synaptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain of the Cav2.1 α1 subunit, most notably to a consensus CaM-binding IQ-like (IQ) domain. Closely related Cav2.2 (N-type) channels display CDI but not CDF, despite overall conservation of the IQ and additional sites (pre-IQ, EF-hand-like [EF] domain, and CaM-binding domain) that regulate CDF of Cav2.1. Here we investigate the molecular determinants that prevent Cav2.2 channels from undergoing CDF. Although alternative splicing of C-terminal exons regulates CDF of Cav2.1, the splicing of analogous exons in Cav2.2 does not reveal CDF. Transfer of sequences encoding the Cav2.1 EF, pre-IQ, and IQ together (EF-pre-IQ-IQ), but not individually, are sufficient to support CDF in chimeric Cav2.2 channels; Cav2.1 chimeras containing the corresponding domains of Cav2.2, either alone or together, fail to undergo CDF. In contrast to the weak binding of CaM to just the pre-IQ and IQ of Cav2.2, CaM binds to the EF-pre-IQ-IQ of Cav2.2 as well as to the corresponding domains of Cav2.1. Therefore, the lack of CDF in Cav2.2 likely arises from an inability of its EF-pre-IQ-IQ to transduce the effects of CaM rather than weak binding to CaM per se. Our results reveal a functional divergence in the CDF regulatory domains of Cav2 channels, which may help to diversify the modes by which Cav2.1 and Cav2.2 can modify synaptic transmission.

Testing the immunocompetence handicap hypothesis: Testosterone manipulation does not affect wound healing in male salamanders.

In vertebrates, a bidirectional relationship exists between the immune system and the hypothalamic-pituitary-gonadal axis. In addition, sexual dimorphism in immunity has been documented in many vertebrates as well as some invertebrates, and males are generally less immunocompetent than their female counterparts. A possible explanation for this is described by the immunocompetence handicap hypothesis (ICHH), which proposes that elevated testosterone (T) levels direct resources towards the promotion of secondary sexual characteristics at a cost to immune function. To further test the ICHH, we examined the effects of T on cutaneous wound healing, an integrative measure of immunity, using male Allegheny Mountain dusky salamanders; a species that has sexually dimorphic courtship glands and testosterone-dependent mating behavior. We did this via two methods: surgical manipulation and transdermal delivery of T. In both experiments, elevated plasma T did not delay wound healing. Interestingly, intact animals healed more slowly than animals that had undergone prior invasive surgery, suggesting that the prior surgery had an immune-priming effect that enhanced healing of a second wound.

Testing hypotheses about individual variation in plasma corticosterone in free-living salamanders.

In vertebrates, many responses to stress as well as homeostatic maintenance of basal metabolism are regulated by plasma glucocorticoid hormones (GCs). Despite having crucial functions, levels of GCs are typically variable among individuals. We examined the contribution of several physiological factors to individual variation in plasma corticosterone (CORT) and the number of corticotropin-releasing hormone (CRH) neurons in the magnocellular preoptic area of the brain in free-living Allegheny Mountain dusky salamanders. We addressed three hypotheses: the current-condition hypothesis, the facilitation hypothesis and the trade-off hypothesis. Differential white blood cell count was identified as a strong contributor to individual variation in baseline CORT, stress-induced CORT and the number of CRH neurons. In contrast, we found no relationship between CORT (or CRH) and body condition, energy stores or reproductive investment, providing no support for the current-condition hypothesis or the trade-off hypothesis involving reproduction. Because of the difficulties of interpreting the functional consequences of variation in differential white blood cell counts, we were unable to distinguish between the facilitation hypothesis or the trade-off hypothesis related to immune function. However, the strong association between differential white blood cell count and hypothalamic-pituitary-adrenal/interrenal (HPA/I) activation suggests that a more thorough examination of immune profiles is critical to understanding variation in HPA/I activation.

Measuring Ca2+-Dependent Modulation of Voltage-Gated Ca2+ Channels in HEK-293T Cells.

Voltage-gated Ca(2+) (Cav) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Cav channels are subject to diverse forms of regulation, including those involving the Ca(2+) ions that permeate the pore. High voltage-activated Cav channels undergo Ca(2+)-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Cav1 (L-type) and Cav2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Cav channel. HEK-293T cells do not express endogenous Cav channels, but Cav channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca(2+)-dependent modulation of recombinant Cav channels in HEK-293T cells.

Treatment with corticosterone delays cutaneous wound healing in male and female salamanders.

In vertebrates, exposure to stressors and stress hormones has a number of physiological effects including modulation of immune function. These effects on immune function have been well studied in mammals, but less is known in other groups, in particular amphibians. To analyze the effects of exposure to stressors and the stress hormone corticosterone, we monitored cutaneous wound healing as a measure of integrated immunity in male and female semi-terrestrial salamanders (Desmognathus ochrophaeus) that were chased to induce endogenous release of corticosterone or were treated with physiologically relevant doses of corticosterone. As predicted, subjects treated daily with corticosterone healed more slowly than did controls. In contrast, subjects that had been chased daily healed at the same rate as controls. Surprisingly, repeated chasing did not elevate plasma corticosterone despite causing drops in body mass and survival. Additionally, females healed more slowly than males, possibly due to energetic constraints.