Functional insights into modulation of BKCa channel activity to alter myometrial contractility

The large-conductance voltage- and Ca2+-activated K+ channel (BKCa) is an important regulator of membrane excitability in a wide variety of cells and tissues. In myometrial smooth muscle, activation of BKCa plays essential roles in buffering contractility to maintain uterine quiescence during pregnancy and in the transition to a more contractile state at the onset of labor. Multiple mechanisms of modulation have been described to alter BKCa channel activity, expression, and cellular localization. In the myometrium, BKCa is regulated by alternative splicing, protein targeting to the plasma membrane, compartmentation in membrane microdomains, and posttranslational modifications. In addition, interaction with auxiliary proteins (i.e., β1- and β2-subunits), association with G-protein coupled receptor signaling pathways, such as those activated by adrenergic and oxytocin receptors, and hormonal regulation provide further mechanisms of variable modulation of BKCa channel function in myometrial smooth muscle. Here, we provide an overview of these mechanisms of BKCa channel modulation and provide a context for them in relation to myometrial function

The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

Although the physiological function of the cellular prion protein (PrPC) remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP)-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+

Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue Virus Serotypes 1 and 8 in a Subclinical Experimental Infection

Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication

Potassium Channels in the Uterine Vasculature: Role in Healthy and Complicated Pregnancies

A progressive increase in maternal uterine and placental blood flow must occur during pregnancy to sustain the development of the fetus. Changes in maternal vasculature enable an increased uterine blood flow, placental nutrient and oxygen exchange, and subsequent fetal development. K+ channels are important modulators of vascular function, promoting vasodilation, inducing cell proliferation, and regulating cell signaling. Different types of K+ channels, such as Ca2+-activated, ATP-sensitive, and voltage-gated, have been implicated in the adaptation of maternal vasculature during pregnancy. Conversely, K+ channel dysfunction has been associated with vascular-related complications of pregnancy, including intrauterine growth restriction and pre-eclampsia. In this article, we provide an updated and comprehensive literature review that highlights the relevance of K+ channels as regulators of uterine vascular reactivity and their potential as therapeutic targets

Effect of β2ND,β1β2β1 and β2NDβ1β2 constructs on the open dwell-times of BK_Ca channels with distinctive N-terminal regions.

<p>Effect of β2ND,β1β2β1 and β2NDβ1β2 constructs on the open dwell-times of BK_Ca channels with distinctive N-terminal regions.</p

BK_Ca α-subunit with distinct N-termini are differentially modulated by β1-β2 chimeric constructs.

<p>Representative inside-out single-channel recordings from HEK293T cells transfected with MANG (<b>A</b>), MSSN (<b>D</b>) or MDAL (<b>G</b>) in the presence or absence of β1β2β1 or β2NDβ1β2, at different membrane potentials (-40 mV to +40 mV) with 10 μM Ca²⁺ in the bath. Dashed lines indicate closed (C) states of the channels. Voltage-activation of MANG (<b>B</b> and <b>C</b>), MSSN (<b>E</b> and <b>F</b>) or MDAL (<b>H</b> and <b>I</b>), in the absence (α, black symbols) or presence of β1 (blue symbols), β2ND (red symbols), β1β2β1 (orange symbols) or β2NDβ1β2 (purple symbols), expressed as open probability (<i>P</i>_<i>o</i>) of the channel, in the presence of 10 μM (<b>B</b>, <b>E</b> and <b>H</b>) or 100 μM (<b>C</b>, <b>F</b>, and <b>I</b>) Ca²⁺ in the bath; n = 3–19, symbols are mean ± SEM.</p

Effects of β2ND and β1-β2 chimeric constructs on BK_Ca α-subunit N-terminal constructs single-channel kinetics.

<p>Open (<b>A</b>) and (<b>B</b>) closed dwell-times distribution histograms of single-channels in HEK293T cells expressing MANG, MSSN or MDAL constructs. Patches containing channels composed by α-subunit alone (black lines), α+β2ND (red lines) α+β1β2β1 (orange lines) or α+β2β1β2 (purple lines) were analyzed (n = 6–11). Single-channel currents were elicited by holding the membrane potential at a certain voltage (-20 mV to -100 mV) for at least 1 min in the presence of 10 μM Ca²⁺ in the bath. Histograms were plotted in log-bin timescales and fitted with double exponential functions.</p

Modulation of voltage-dependent activation in BK_Ca α-subunit with distinct N-termini by β-subunit constructs.

<p>Analysis of the voltage of half maximal activation (<i>V</i>_{<i>0</i>.<i>5</i>}) of the N-terminal BK_Ca constructs MANG (<b>A</b>), MSSN (<b>B</b>) or MDAL (<b>C</b>), in the absence (α, black symbols) or presence of β1 (blue symbols), β2ND (red symbols), β1β2β1 (orange symbols) or β2NDβ1β2 (purple symbols), at different [Ca²⁺] in the bath. Symbols are mean ± SEM.</p

The unique N-terminal sequence of the BK_Ca channel α-subunit determines its modulation by β-subunits

<div><p>Large conductance voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels are essential regulators of membrane excitability in a wide variety of cells and tissues. An important mechanism of modulation of BK_Ca channel activity is its association with auxiliary subunits. In smooth muscle cells, the most predominant regulatory subunit of BK_Ca channels is the β1-subunit. We have previously described that BK_Ca channels with distinctive N-terminal ends (starting with the amino acid sequence MDAL, MSSN or MANG) are differentially modulated by the β1-subunit, but not by the β2. Here we extended our studies to understand how the distinct N-terminal regions differentially modulate channel activity by β-subunits. We recorded inside-out single-channel currents from HEK293T cells co-expressing the BK_Ca containing three N-terminal sequences with two β1-β2 chimeric constructs containing the extracellular loop of β1 or β2, and the transmembrane and cytoplasmic domains of β2 or β1, respectively. Both β chimeric constructs induced leftward shifts of voltage-activation curves of channels starting with MANG and MDAL, in the presence of 10 or 100 μM intracellular Ca²⁺. However, MSSN showed no shift of the voltage-activation, at the same Ca²⁺ concentrations. The presence of the extracellular loop of β1 in the chimera resembled results seen with the full β1 subunit, suggesting that the extracellular region of β1 might be responsible for the lack of modulation observed in MSSN. We further studied a poly-serine stretch present in the N-terminal region of MSSN and observed that the voltage-activation curves of BK_Ca channels either containing or lacking this poly-serine stretch were leftward shifted by β1-subunit in a similar way. Overall, our results provide further insights into the mechanism of modulation of the different N-terminal regions of the BK_Ca channel by β-subunits and highlight the extension of this region of the channel as a form of modulation of channel activity.</p></div