Oana N. Ureche

Regulation of the Glutamate Transporter EAAT4 by PIKfyve

The excitatory amino-acid transporter EAAT4 (SLC1A6), a Na+,glutamate cotransporter expressed mainly in Purkinje cells, serves to clear glutamate from the synaptic cleft. EAAT4 activity is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent regulation of the Na+,glucose transporter SGLT1 (SLC5A1) and the creatine transporter CreaT (SLC6A8) has recently been shown to involve the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether SGK1-dependent EAAT4-regulation similarly involves PIKfyve. In Xenopus oocytes expressing EAAT4, but not in water injected oocytes, glutamate induced a current which was significantly enhanced by coexpression of PIKfyve and SGK1. The glutamate induced current in Xenopus oocytes coexpressing EAAT4 and both, PIKfyve and SGK1, was significantly larger than the current in Xenopus oocytes expressing EAAT4 together with either kinase alone. Coexpression of the inactive SGK1 mutant K127NSGK1 did not significantly alter glutamate induced current in EAAT4-expressing Xenopus oocytes and abolished the stimulation of glutamate induced current by coexpression of PIKfyve. The stimulating effect of PIKfyve was abrogated by replacement of the serine with alanine in the SGK consensus sequence (S318APIKfyve). Furthermore, coexpression of S318APIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.

Long QT Syndrome–Associated Mutations in KCNQ1 and KCNE1 Subunits Disrupt Normal Endosomal Recycling of IKs Channels

Physical and emotional stress is accompanied by release of stress hormones such as the glucocorticoid cortisol. This hormone upregulates the serum- and glucocorticoid-inducible kinase (SGK)1, which in turn stimulates IKs, a slow delayed rectifier potassium current that mediates cardiac action potential repolarization. Mutations in IKs channel &agr; (KCNQ1, KvLQT1, Kv7.1) or &bgr; (KCNE1, IsK, minK) subunits cause long QT syndrome (LQTS), an inherited cardiac arrhythmia associated with increased risk of sudden death. Together with the GTPases RAB5 and RAB11, SGK1 facilitates membrane recycling of KCNQ1 channels. Here, we show altered SGK1-dependent regulation of LQTS-associated mutant IKs channels. Whereas some mutant KCNQ1 channels had reduced basal activity but were still activated by SGK1, currents mediated by KCNQ1(Y111C) or KCNQ1(L114P) were paradoxically reduced by SGK1. Heteromeric channels coassembled of wild-type KCNQ1 and the LQTS-associated KCNE1(D76N) mutant were similarly downregulated by SGK1 because of a disrupted RAB11-dependent recycling. Mutagenesis experiments indicate that stimulation of IKs channels by SGK1 depends on residues H73, N75, D76, and P77 in KCNE1. Identification of the IKs recycling pathway and its modulation by stress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered by physical or psychological stress.

A schizophrenia-linked mutation in PIP5K2A fails to activate neuronal M channels

RationaleEvidence for an association between phosphatidylinositol-4-phosphate 5-kinase II alpha (PIP5K2A) and schizophrenia was recently obtained and replicated in several samples. PIP5K2A controls the function of KCNQ channels via phosphatidylinositol-4,5-bisphosphate (PIP2) synthesis. Interestingly, recent data suggest that KCNQ channels suppress basal activity of dopaminergic neurons and dopaminergic firing. Activation of KCNQ accordingly attenuates the central stimulating effects of dopamine, cocaine, methylphenidate, and phenylcyclidine.ObjectiveThe aim of this study was to explore the functional relevance of PIP5K2A, which might influence schizophrenic behavior.Materials and methodsHere, we study the effects of the neuronal PIP5K2A on KCNQ2, KCNQ5, KCNQ2/KCNQ3, and KCNQ3/KCNQ5 in the Xenopus expression system.ResultsWe find that wild-type PIP5K2A but not the schizophrenia-associated mutant (N251S)-PIP5K2A activates heteromeric KCNQ2/KCNQ3 and KCNQ3/KCNQ5, the molecular correlate of neuronal M channels. Homomeric KCNQ2 and KCNQ5 channels were not activated by the kinase indicating that the presence of KCNQ3 in the channel complex is required for the kinase-mediated effects. Acute application of PI(4,5)P2 and a PIP2 scavenger indicates that the mutation N251S renders the kinase PIP5K2A inactive.ConclusionsOur results suggest that the schizophrenia-linked mutation of the kinase results in reduced KCNQ channel function and thereby might explain the loss of dopaminergic control in schizophrenic patients. Moreover, the addictive potential of dopaminergic drugs often observed in schizophrenic patients might be explained by this mechanism. At least, the insufficiency of (N251S)-PIP5K2A to stimulate neuronal M channels may contribute to the clinical phenotype of schizophrenia.

Functional significance of the kainate receptor GluR6(M836I) mutation that is linked to autism.

Previous studies revealed a linkage of the kainate receptor GluR6 with autism, a pervasive developmental disorder. Mutational screening in autistic patients disclosed the amino acid exchange M836I in a highly conserved domain of the cytoplasmic C-terminal region of GluR6. Here, we show that this mutation leads to GluR6 gain-of-function. By using the two-electrode voltage clamp technique we observed a significant increase of current amplitudes of mutant GluR6 compared to wild type GluR6. Western blotting of oocytes injected with mutant or wild type GluR6 cRNA and transfection of EGFP-tagged GluR6 receptors into COS-7 cells revealed an enhanced plasma membrane expression of GluR6(M836I) compared to wild type GluR6. Membrane expression of GluR6(M836I) but not of wild type GluR6 seems to be regulated by Rab11 as indicated by our finding that GluR6(M836I) but not wild type GluR6 showed increased current amplitudes and protein expression when coexpressed with Rab11. Furthermore, injection of GTP plus Rab11A protein into oocytes increased current amplitudes in GluR6(M836I) but not in wild type GluR6. By contrast, Rab5 downregulated the currents in oocytes expressing wild type GluR6 but had only little, statistically not significant effects on currents in oocytes expressing GluR6(M836I). Our data on altered functional properties of GluR6(M836I) provide a functional basis for the postulated linkage of GluR6 to autism. Furthermore, we identified new mechanisms determining the plasma membrane abundance of wild type GluR6 and GluR6(M836I).

PIP5K2A-dependent regulation of excitatory amino acid transporter EAAT3

IntroductionAccording to previous observations, the gene encoding the phosphatidylinositol-4-phosphate 5-kinase II alpha (PIP5K2A) is associated with schizophrenia. Specifically, the mutation N251SPIP5K2A has been discovered in schizophrenic patients but not in healthy individuals. A defect of the excitatory amino acid transporter EAAT3 has similarly been implicated in the development of schizophrenia. The present study thus explored whether PIP5K2A is involved in the regulation of EAAT3 activity.Materials and methodsEAAT3 was expressed in Xenopus oocytes either without or with PIP5K2A, and EAAT3 transporter activity was estimated from the glutamate (2-mM)-induced current (Iglu) in dual electrode voltage clamp experiments. EAAT3 protein abundance in the cell membrane was estimated by Western blotting and confocal microscopy.ResultsIn EAAT3-expressing oocytes, Iglu was enhanced by coexpression of wild type PIP5K2A. Coexpression of the schizophrenia-associated mutant N251SPIP5K2A significantly decreased Iglu in oocytes expressing EAAT3 with or without additional expression of wild type PIP5K2A. Thus, N251SPIP5K2A exerts a dominant inhibitory effect.DiscussionMembrane abundance of EAAT3 was increased by wild type PIP5K2A and decreased by N251SPIP5K2A in both EAAT3-expressing oocytes and human embryonic kidney cells. The present observations disclose a novel mechanism of EAAT3 regulation, which may contribute to the deranged regulation of excitability in schizophrenic patients.

Novel Insights into the Structural Basis of pH-Sensitivity in Inward Rectifier K+ Channels Kir2.3

The Kir2 channels belong to a family of potassium selective channels with characteristic strong inward rectification. Heteromeric assemblies of Kir2.1, Kir2.2 and Kir2.3 channels underly membrane potential stabilizing currents in ventricular myocytes, neurons and skeletal muscle. Kir2 channels differ substantially in their sensitivity to extracellular pH. The extracellular histidine Kir2.3(H117) contributes to the pH dependence of K-channels containing Kir2.3. Here, we study the possibility of intramolecular interactions of the residue Kir2.3(H117) with conserved cysteines in close proximity to the selectivity filter. We engineered a cobalt coordination site and reduction/oxidation sensitivity in Kir2.3 by introduction of a cysteine into the putatively hydrogen bonding residue (Kir2.3(H117C)) confirming that this residue is in proximity to Kir2.3(C141). Using SCAM we determined the location of the Kir2.3(H117) in the outer pore mouth and incorporated these data into a 3D model. We conclude that formation of a hydrogen bond at low pH may stabilize the outer pore domain to favour the selectivity filter in a slightly distorted conformation thus reducing ion permeation. The data provide molecular insight into the unique pH regulation of inward rectifier channels.

PIKfyve upregulates CFTR activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel critically important in Cl(-) secreting epithelia. Mutations in the CFTR gene, such as (DeltaF508)CFTR leads to cystic fibrosis, a severe disease with defective Cl(-) secretion. CFTR is stimulated by the serum and glucocorticoid-inducible kinase SGK1. The SGK1 dependent regulation of several carriers and channels involves the phosphatidylinositol-3-phosphate-5-kinase PIKfyve, which similarly mediates the regulation of glucose carriers by PKB/Akt. The present study was thus performed to elucidate whether PKB/Akt and PIKfyve are regulators of CFTR. To this end CFTR or (DeltaF508)CFTR were expressed in Xenopus oocytes alone or together with PKB, PIKfyve or the SGK1/PKB resistant mutant (S318A)PIKfyve, and the current generated by cAMP upregulation with 10muM forskolin+1mM IBMX determined utilizing dual electrode voltage clamp. As a result, forskolin/IBMX treatment triggered a current (I(cAMP)) in CFTR-expressing Xenopus oocytes, but not in oocytes expressing (DeltaF508)CFTR. Coexpression of PKB/Akt and PIKfyve, but not of (S318A)PIKfyve, stimulated I(cAMP) in CFTR-expressing ( approximately 2- to 3-fold) but not in (DeltaF508)CFTR-expressing or water injected Xenopus oocytes. Immunohistochemistry revealed that the coexpression of PIKfyve, but not of (S318A)PIKfyve, enhanced the CFTR protein abundance but not the (DeltaF508)CFTR protein abundance in CFTR or (DeltaF508)CFTR-expressing oocytes. The present observations reveal a novel powerful regulator of intact but not of defective CFTR.

Differential modulation of cardiac potassium channels by Grb adaptor proteins

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K(+) channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K(+) channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K(+) channels.

293 – Impaired function of schizophrenia associated mutant PIP5K2A

Background: Schizophrenia has been called a cruel disease that impairs life greatly in a degenerative fashion, altering emotions and various abilities greatly. Many patients are treated with anticholinergic agents that are known to modulate phosphoinositide metabolism. Not surprisingly, a number of genes involved in the synthesis or dephosphorylation of PI(4,5)P2 map very closely to regions of the genome that have been linked to schizophrenia. These include the phosphatidylinositol 3-kinases and PIP5K2A, a member of the phosphatidylinositol 4-phosphate 5-kinase family and recently it has been found that the mutation (N251S)–PIP5K2A could be genetically linked to schizophrenia. M-channels, voltage-gated K+ channels that regulate the excitability of many neurons are regulated by the product PIP2 of PIP5K2A. Neuronal Kv7.2/Kv7.3 and Kv7.3/Kv7.5 channels require PIP2 to open. Here, we study the effects of the neuronal PIP5K2A on KCNQ2, KCNQ5, KCNQ2/KCNQ3 and KCNQ3/ KCNQ5 in the Xenopus oocytes expression system. Methods: Electrophysiology-Standard two-electrode voltage-clamp techniques were used to record whole cell currents in Xenopus oocytes at room temperature. KCNQ2, KCNQ3 and KCNQ5 were subcloned in oocyte expression vector pSGEM. All constructs were confirmed by automated DNA sequencing. Results: We found that wild-type PIP5K2A but not the schizophrenia associated mutant (N251S)–PIP5K2A activates heteromeric neuronal M-channels KCNQ2/KCNQ3 and KCNQ3/KCNQ5. Homomeric KCNQ2 and KCNQ5 channels were not activated by the kinase indicating that the presence of KCNQ3 in the channel complex is required for the kinase mediated effects. The mutation in the kinase may result in structural changes in the catalytic domain and nonfunctionality. Conclusions: Thus, the insufficiency to stimulate PIP2 of (N251S)– PIP5K2A may contribute to the clinical phenotype of schizophrenia. Acknowledgement: The work of Dr. Olga Fedorenko was supported by INTAS YS Fellowship (Ref. N. 04-83-3764).