Functional analysis of missense variants in the TRESK (KCNK18) K+ channel

A loss of function mutation in the TRESK K2P potassium channel (KCNK18), has recently been linked with typical familial migraine with aura. We now report the functional characterisation of additional TRESK channel missense variants identified in unrelated patients. Several variants either had no apparent functional effect, or they caused a reduction in channel activity. However, the C110R variant was found to cause a complete loss of TRESK function, yet is present in both sporadic migraine and control cohorts, and no variation in KCNK18 copy number was found. Thus despite the previously identified association between loss of TRESK channel activity and migraine in a large multigenerational pedigree, this finding indicates that a single non-functional TRESK variant is not alone sufficient to cause typical migraine and highlights the genetic complexity of this disorder

Functional kinomics establishes a critical node of volume-sensitive cation-Cl^- cotransporter regulation in the mammalian brain

This is the final version of the article. Available from the publisher via the DOI in this record.There is another record in ORE for this publication: http://hdl.handle.net/10871/33424Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation – a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl−-sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl− uptake and stimulation of KCC3-mediated Cl− extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought “Cl−/volume-sensitive kinase” of the cation-Cl− cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.We thank the excellent technical support of the MRC-Protein Phosphorylation and Ubiquitylation Unit (PPU) DNA Sequencing Service (coordinated by Nicholas Helps), the MRC-PPU tissue culture team (coordinated by Laura Fin), the Division of Signal Transduction Therapy (DSTT) antibody purification teams (coordinated by Hilary McLauchlan and James Hastie). We are grateful to the MRC PPU Proteomics facility (coordinated by David Campbell, Robert Gourlay and Joby Varghese). We thank for support the Medical Research Council (MC_UU_12016/2; DRA) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer; DRA). We thank Thomas J. Jentsch (Max-Delbrück-Centrum für Molekulare Medizin) for providing the KCC1/3 double KO mice and his reading of this manuscript. We thank Nathaniel Grey (Harvard) for providing the kinase inhibitor library used in this study (NIH LINCS Program grant U54HL127365). This work was also supported by a Harvard-MIT Neuroscience Grant (to KTK/SJE)

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro.

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell

Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating

The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel’s selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating

Phosphorylation and Transport in the Na-K-2Cl Cotransporters, NKCC1 and NKCC2A, Compared in HEK-293 Cells

Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates

Differentiated Human Midbrain-Derived Neural Progenitor Cells Express Excitatory Strychnine-Sensitive Glycine Receptors Containing α2β Subunits

BACKGROUND: Human fetal midbrain-derived neural progenitor cells (NPCs) may deliver a tissue source for drug screening and regenerative cell therapy to treat Parkinson's disease. While glutamate and GABA(A) receptors play an important role in neurogenesis, the involvement of glycine receptors during human neurogenesis and dopaminergic differentiation as well as their molecular and functional characteristics in NPCs are largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigated NPCs in respect to their glycine receptor function and subunit expression using electrophysiology, calcium imaging, immunocytochemistry, and quantitative real-time PCR. Whole-cell recordings demonstrate the ability of NPCs to express functional strychnine-sensitive glycine receptors after differentiation for 3 weeks in vitro. Pharmacological and molecular analyses indicate a predominance of glycine receptor heteromers containing α2β subunits. Intracellular calcium measurements of differentiated NPCs suggest that glycine evokes depolarisations mediated by strychnine-sensitive glycine receptors and not by D-serine-sensitive excitatory glycine receptors. Culturing NPCs with additional glycine, the glycine-receptor antagonist strychnine, or the Na(+)-K(+)-Cl(-) co-transporter 1 (NKCC1)-inhibitor bumetanide did not significantly influence cell proliferation and differentiation in vitro. CONCLUSIONS/SIGNIFICANCE: These data indicate that NPCs derived from human fetal midbrain tissue acquire essential glycine receptor properties during neuronal maturation. However, glycine receptors seem to have a limited functional impact on neurogenesis and dopaminergic differentiation of NPCs in vitro

Aldosterone does not require angiotensin II to activate NCC through a WNK4–SPAK–dependent pathway

We and others have recently shown that angiotensin II can activate the sodium chloride cotransporter (NCC) through a WNK4–SPAK-dependent pathway. Because WNK4 was previously shown to be a negative regulator of NCC, it has been postulated that angiotensin II converts WNK4 to a positive regulator. Here, we ask whether aldosterone requires angiotensin II to activate NCC and if their effects are additive. To do so, we infused vehicle or aldosterone in adrenalectomized rats that also received the angiotensin receptor blocker losartan. In the presence of losartan, aldosterone was still capable of increasing total and phosphorylated NCC twofold to threefold. The kinases WNK4 and SPAK also increased with aldosterone and losartan. A dose-dependent relationship between aldosterone and NCC, SPAK, and WNK4 was identified, suggesting that these are aldosterone-sensitive proteins. As more functional evidence of increased NCC activity, we showed that rats receiving aldosterone and losartan had a significantly greater natriuretic response to hydrochlorothiazide than rats receiving losartan only. To study whether angiotensin II could have an additive effect, rats receiving aldosterone with losartan were compared with rats receiving aldosterone only. Rats receiving aldosterone only retained more sodium and had twofold to fourfold increase in phosphorylated NCC. Together, our results demonstrate that aldosterone does not require angiotensin II to activate NCC and that WNK4 appears to act as a positive regulator in this pathway. The additive effect of angiotensin II may favor electroneutral sodium reabsorption during hypovolemia and may contribute to hypertension in diseases with an activated renin–angiotensin–aldosterone system

Analysis of Adhesion Molecules and Basement Membrane Contributions to Synaptic Adhesion at the Drosophila Embryonic NMJ

Synapse formation and maintenance crucially underlie brain function in health and disease. Both processes are believed to depend on cell adhesion molecules (CAMs). Many different classes of CAMs localise to synapses, including cadherins, protocadherins, neuroligins, neurexins, integrins, and immunoglobulin adhesion proteins, and further contributions come from the extracellular matrix and its receptors. Most of these factors have been scrutinised by loss-of-function analyses in animal models. However, which adhesion factors establish the essential physical links across synaptic clefts and allow the assembly of synaptic machineries at the contact site in vivo is still unclear. To investigate these key questions, we have used the neuromuscular junction (NMJ) of Drosophila embryos as a genetically amenable model synapse. Our ultrastructural analyses of NMJs lacking different classes of CAMs revealed that loss of all neurexins, all classical cadherins or all glutamate receptors, as well as combinations between these or with a Laminin deficiency, failed to reveal structural phenotypes. These results are compatible with a view that these CAMs might have no structural role at this model synapse. However, we consider it far more likely that they operate in a redundant or well buffered context. We propose a model based on a multi-adaptor principle to explain this phenomenon. Furthermore, we report a new CAM-independent adhesion mechanism that involves the basement membranes (BM) covering neuromuscular terminals. Thus, motorneuronal terminals show strong partial detachment of the junction when BM-to-cell surface attachment is impaired by removing Laminin A, or when BMs lose their structural integrity upon loss of type IV collagens. We conclude that BMs are essential to tie embryonic motorneuronal terminals to the muscle surface, lending CAM-independent structural support to their adhesion. Therefore, future developmental studies of these synaptic junctions in Drosophila need to consider the important contribution made by BM-dependent mechanisms, in addition to CAM-dependent adhesion

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function