The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1 : contributions of the pore domains

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb+ or Na+ replaced extracellular K+. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels nonselective. We interpret the effects of these—and of other mutations—in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter

Contesting the Dominant Discourse of Child Sexual Abuse: Sexual Subjects, Agency, and Ethics

Responding to previous scholars’ call to explore the complexities of child sexual abuse (CSA), this article presents narratives of CSA and scrutinizes a binary construction underpinning this discourse of CSA, namely, the positioning of children as powerless and adults as powerful. The narratives belong to three Indonesian young people who have had sexual interactions with adults when they were children. The findings demonstrate how this binary positioning has been both drawn upon and resisted in the ways participants understand their sexual experiences. This article contributes to the existing literature by providing analyses of some vignettes of everyday experiences of how children might be constituted as sexual subjects, including their capability to exercise agency, perform resistance, and negotiate ethics. The implications of the findings are discussed in relation to how the recognition of children as sexual subjects and their sexual agency might be beneficial for parents, educators, and counselors

Gating of a pH-Sensitive K2P Potassium Channel by an Electrostatic Effect of Basic Sensor Residues on the Selectivity Filter

K+ channels share common selectivity characteristics but exhibit a wide diversity in how they are gated open. Leak K2P K+ channels TASK-2, TALK-1 and TALK-2 are gated open by extracellular alkalinization. The mechanism for this alkalinization-dependent gating has been proposed to be the neutralization of the side chain of a single arginine (lysine in TALK-2) residue near the pore of TASK-2, which occurs with the unusual pKa of 8.0. We now corroborate this hypothesis by transplanting the TASK-2 extracellular pH (pHo) sensor in the background of a pHo-insensitive TASK-3 channel, which leads to the restitution of pHo-gating. Using a concatenated channel approach, we also demonstrate that for TASK-2 to open, pHo sensors must be neutralized in each of the two subunits forming these dimeric channels with no apparent cross-talk between the sensors. These results are consistent with adaptive biasing force analysis of K+ permeation using a model selectivity filter in wild-type and mutated channels. The underlying free-energy profiles confirm that either a doubly or a singly charged pHo sensor is sufficient to abolish ion flow. Atomic detail of the associated mechanism reveals that, rather than a collapse of the pore, as proposed for other K2P channels gated at the selectivity filter, an increased height of the energetic barriers for ion translocation accounts for channel blockade at acid pHo. Our data, therefore, strongly suggest that a cycle of protonation/deprotonation of pHo-sensing arginine 224 side chain gates the TASK-2 channel by electrostatically tuning the conformational stability of its selectivity filter

Social anxiety symptoms in young children:Investigating the interplay of theory of mind and expressions of shyness

Children’s early onset of social anxiety may be associated with their social understanding, and their ability to express emotions adaptively. We examined whether social anxiety in 48-month-old children (N = 110; 54 boys) was related to: a) a lower level of theory of mind (ToM); b) a lower proclivity to express shyness in a positive way (adaptive); and c) a higher tendency to express shyness in a negative way (non-adaptive). In addition, we investigated to what extent children’s level of social anxiety was predicted by the interaction between ToM and expressions of shyness. Children’s positive and negative expressions of shyness were observed during a performance task. ToM was measured with a validated battery, and social anxiety was assessed using both parents’ reports on questionnaires. Socially anxious children had a lower level of ToM, and displayed more negative and less positive shy expressions. However, children with a lower level of ToM who expressed more positive shyness were less socially anxious. Additional results show that children who displayed shyness only in a negative manner were more socially anxious than children who expressed shyness only in a positive way and children who did not display any shyness. Moreover, children who displayed both positive and negative expressions of shyness were more socially anxious than children who displayed shyness only in a positive way. These findings highlight the importance of ToM development and socio-emotional strategies, and their interaction, on the early development of social anxiety

Prokineticin 2 Regulates the Electrical Activity of Rat Suprachiasmatic Nuclei Neurons

Neuropeptide signaling plays roles in coordinating cellular activities and maintaining robust oscillations within the mammalian suprachiasmatic nucleus (SCN). Prokineticin2 (PK2) is a signaling molecule from the SCN and involves in the generation of circadian locomotor activity. Prokineticin receptor 2 (PKR2), a receptor for PK2, has been shown to be expressed in the SCN. However, very little is known about the cellular action of PK2 within the SCN. In the present study, we investigated the effect of PK2 on spontaneous firing and miniature inhibitory postsynaptic currents (mIPSCs) using whole cell patch-clamp recording in the SCN slices. PK2 dose-dependently increased spontaneous firing rates in most neurons from the dorsal SCN. PK2 acted postsynaptically to reduce γ-aminobutyric acid (GABA)-ergic function within the SCN, and PK2 reduced the amplitude but not frequency of mIPSCs. Furthermore, PK2 also suppressed exogenous GABA-induced currents. And the inhibitory effect of PK2 required PKC activation in the postsynaptic cells. Our data suggest that PK2 could alter cellular activities within the SCN and may influence behavioral and physiological rhythms

Smooth muscle Ca(2+) -activated and voltage-gated K+ channels modulate conducted dilation in rat isolated small mesenteric arteries.

OBJECTIVE:   To assess the influence of blocking smooth muscle large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels on the conducted dilation to ACh and isoproterenol. MATERIALS AND METHODS:   Rat mesenteric arteries were isolated with a bifurcation, triple-cannulated, pressurized and imaged using confocal microscopy. Phenylephrine was added to the superfusate to generate tone, and agonists perfused into a sidebranch to evoke local dilation and subsequent conducted dilation into the feed artery. RESULTS:   Both ACh- and isoproterenol-stimulated local and conducted dilation with similar magnitudes of decay with distance along the feed artery (2000μm: ∼15% maximum dilation). The gap junction uncoupler carbenoxolone prevented both conducted dilation and intercellular spread of dye through gap junctions. IbTx, TEA or 4-AP, blockers of large conductance Ca(2+) -activated K+ channels and voltage-gated K+ channels, did not affect conducted dilation to either agonist. A combination of either IbTx or TEA with 4-AP markedly improved the extent of conducted dilation to both agonists (2000μm: >50% maximum dilation). The enhanced conducted dilation was reflected in the hyperpolarization to ACh (2000μm: Control, 4±1 mV, n = 3; TEA with 4-AP, 14±3mV, n=4), and was dependent on the endothelium. CONCLUSIONS:   These data show that activated BK(Ca) and K(V) -channels serve to reduce the effectiveness of conducted dilation

Nitric oxide suppresses cerebral vasomotion by sGC-independent effects on ryanodine receptors and voltage-gated calcium channels.

BACKGROUND/AIMS: In cerebral arteries, nitric oxide (NO) release plays a key role in suppressing vasomotion. Our aim was to establish the pathways affected by NO in rat middle cerebral arteries. METHODS: In isolated segments of artery, isometric tension and simultaneous measurements of either smooth muscle membrane potential or intracellular [Ca(2+)] ([Ca(2+)](SMC)) changes were recorded. RESULTS: In the absence of L-NAME, asynchronous propagating Ca(2+) waves were recorded that were sensitive to block with ryanodine, but not nifedipine. L-NAME stimulated pronounced vasomotion and synchronous Ca(2+) oscillations with close temporal coupling between membrane potential, tone and [Ca(2+)](SMC). If nifedipine was applied together with L-NAME, [Ca(2+)](SMC) decreased and synchronous Ca(2+) oscillations were lost, but asynchronous propagating Ca(2+) waves persisted. Vasomotion was similarly evoked by either iberiotoxin, or by ryanodine, and to a lesser extent by ODQ. Exogenous application of NONOate stimulated endothelium-independent hyperpolarization and relaxation of either L-NAME-induced or spontaneous arterial tone. NO-evoked hyperpolarization involved activation of BK(Ca) channels via ryanodine receptors (RYRs), with little involvement of sGC. Further, in whole cell mode, NO inhibited current through L-type voltage-gated Ca(2+) channels (VGCC), which was independent of both voltage and sGC. CONCLUSION: NO exerts sGC-independent actions at RYRs and at VGCC, both of which normally suppress cerebral artery myogenic tone

A novel role for HNO in local and spreading vasodilatation in rat mesenteric resistance arteries.

Nitric oxide-mediated vasodilatation has previously been attributed to the uncharged form of the molecule (NO(•)), but increasing evidence suggests that nitroxyl (HNO) may play a significant role in endothelium-dependent relaxation. The aim of this study was to investigate the mechanisms underlying HNO-mediated vasodilatation in phenylephrine pre-constricted pressurized (70 mmHg) mesenteric arteries, and on membrane currents in isolated smooth muscle cells using whole cell and perforated patch clamp recordings. Angeli's salt (AS: nitroxyl donor), evoked concentration-dependent vasodilatation that was insensitive to the NO(•) scavengers carboxy-PTIO and hydroxocobalamin (HXC), but sensitive to either the HNO scavenger L-cysteine, K-channel blockers (4-AP and iberiotoxin), raised [K(+)](o), or inhibition of soluble guanylyl cyclase (ODQ). AS-evoked smooth muscle hyperpolarization significantly augmented K(V) current in an ODQ sensitive manner, and also increased the BK(Ca) current. Importantly, 30 μM AS initiated conducted or spreading vasodilatation, and following blockade of endothelial K-channels (TRAM-34 and apamin), ACh was able to evoke similar L-cysteine-sensitive conducted dilatation. These data show that vasodilatation induced by HNO is mediated by both K(V) and BK(Ca) channels, and suggest a physiological role in vasodilatation through the vasculature