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 I(Ks), a slow delayed rectifier potassium current that mediates cardiac action potential repolarization. Mutations in I(Ks) channel alpha (KCNQ1, KvLQT1, Kv7.1) or beta (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 I(Ks) 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 I(Ks) channels by SGK1 depends on residues H73, N75, D76, and P77 in KCNE1. Identification of the I(Ks) recycling pathway and its modulation by stress-stimulated SGK1 provides novel mechanistic insight into potentially fatal cardiac arrhythmias triggered by physical or psychological stress.

Regulation of TGF-(beta) signalling by N-acetylgalactosaminyltransferase-like 1.

The TGF-beta superfamily of secreted signalling molecules plays a pivotal role in the regulation of early embryogenesis, organogenesis and adult tissue homeostasis. Here we report the identification of Xenopus N-acetylgalactosaminyltransferase-like 1 (xGalntl-1) as a novel important regulator of TGF-beta signalling. N-acetylgalactosaminyltransferases mediate the first step of mucin-type glycosylation, adding N-acetylgalactose to serine or threonine side chains. xGalntl-1 is expressed in the anterior mesoderm and neural crest territory at neurula stage, and in the anterior neural crest, notochord and the mediolateral spinal cord at tailbud stage. Inhibition of endogenous xGalntl-1 protein synthesis, using specific morpholino oligomers, interfered with the formation of anterior neural crest, anterior notochord and the spinal cord. Xenopus and mammalian Galntl-1 inhibited Activin as well as BMP signalling in the early Xenopus embryo and in human HEK 293T cells. Gain- and loss-of-function experiments showed that xGalntl-1 interferes with the activity of the common TGF-beta type II receptor ActR-IIB in vivo. In addition, our biochemical data demonstrated that xGalntl-1 specifically interferes with the binding of ActR-IIB to Activin- and BMP-specific type I receptors. This inhibitory activity of xGalntl-1 was dependent on mucin-type glycosylation, as it was sensitive to the chemical inhibitor benzyl-GalNAc. These studies reveal an important role of a N-acetylgalactosaminyltransferase in the regulation of TGF-beta signalling. This novel regulatory mechanism is evolutionarily conserved and, thus, might provide a new paradigm for the regulation of TGF-beta signalling in vertebrates.

Regulation of endocytic recycling of KCNQ1/KCNE1 potassium channels.

Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, I(Ks). KCNQ1 and KCNE1 subunits coassemble to form the I(Ks) channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death. Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum- and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P(2). Identification of KCNQ1/KCNE1 recycling and its modulation by serum- and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P(2) provides a mechanistic insight into stress-induced acceleration of cardiac repolarization.

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).

Cytoplasmic accumulation of long-chain coenzyme A esters activates KATP and inhibits Kir2.1 channels.

Long-chain fatty acids acyl coenzyme A esters (LC-CoA) are obligate intermediates of fatty acid metabolism and have been shown to activate K(ATP) channels but to inhibit most other Kir channels (e.g. Kir2.1) by direct channel binding. The activation of K(ATP) channels by elevated levels of LC-CoA may be involved in the pathophysiology of type 2 diabetes, the hypothalamic sensing of circulating fatty acids and the regulation of cardiac K(ATP) channels. However, LC-CoA are effectively buffered in the cytoplasm and it is currently not clear whether their free concentration can reach levels sufficient to affect Kir channels in vivo. Here, we report that extracellular oleic acid complexed with albumin at an unbound concentration of 81 +/- 1 nm strongly activated K(ATP) channels and inhibited Kir2.1 channels in Chinese hamster ovary (CHO) cells as well as endogenous Kir currents in human embryonic kidney (HEK293) cells. These effects were only seen in the presence of a high concentration of glucose (25 mm), a condition known to promote the accumulation of LC-CoA by inhibiting their mitochondrial uptake via carnitine-palmitoyl-transferase-1 (CPT1). Accordingly, pharmacological inhibition of CPT1 by etomoxir restored the effects of oleic acid under low glucose conditions. Finally, triacsin C, an inhibitor of the acyl-CoA synthetase, which is necessary for LC-CoA formation, abolished the effects of extracellular oleic acid on the various Kir channels. These results establish the direct regulation of Kir channels by the cytoplasmic accumulation of LC-CoA, which might be of physiological and pathophysiological relevance in a variety of tissues.

Additive regulation of GluR1 by stargazin and serum- and glucocorticoid-inducible kinase isoform SGK3.

The serum- and glucocorticoid-inducible kinase isoform 3 (SGK3) and stargazin have both been shown to enhance the synaptic expression level of GluR1. The present study was performed to elucidate whether SGK3 and stargazin interact or are effective through different pathways in the regulation of GluR1. Proteins were expressed in Xenopus oocytes by injection of complementary RNA (cRNA) encoding GluR1, SGK isoforms, and/or stargazin. In oocytes expressing GluR1 6 days after cRNA injection, glutamate induced an inward current (IGlu), which was increased approximately fourfold following coexpression of SGK3. Coexpression of stargazin similarly enhanced IGlu. Coexpression of both SGK3 and stargazin stimulated the current by a factor of 15.5. Replacement of the serine by alanine at the only SGK consensus sequence (RXRXXS/T) in stargazin enhanced the efficacy of stargazin but did not prevent further stimulation of IGlu by additional coexpression of SGK3. Western blotting showed that stargazin accelerated membrane insertion of GluR1 protein leading to enhanced GluR1 plasma membrane protein abundance 2 days, but not 6 days, after cRNA injection, while SGK3 increased plasma membrane protein abundance 6 days after cRNA injection. In conclusion, SGK3 and stargazin regulate GluR1 independently, and thus, their effects on glutamate-induced currents are additive.

Regulation of KCNQ4 potassium channel prepulse dependence and current amplitude by SGK1 in Xenopus oocytes.

The KCNQ gene family comprises voltage-gated potassium channels expressed in epithelial tissues (KCNQ1, KCNQ5), inner ear structures (KCNQ1, KCNQ4) and the brain (KCNQ2-5). KCNQ4 is expressed in inner and outer hair cells of the inner ear where it determines electrical excitability. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss. Several K+ channels including the closely related KCNQ1/KCNE1 channel are regulated by the serum- and glucocorticoid-inducible kinase (SGK) family. The present study utilized the Xenopus oocyte system to explore effects of SGK isoforms on KCNQ4 mediated K(+)-currents: KCNQ4 channels activated in a voltage dependent manner with half maximal activation at -10 mV. The peak channel activity was significantly increased by prepulsing. Coexpression of wild type SGK1 but not coexpression of the inactive mutant (K127N)SGK1 significantly increased current amplitudes (by 67 %) and significantly increased the resting potential of KCNQ4 expressing oocytes. Here we describe for the first time a prepulse dependence of KCNQ4 channels with increased currents after hyperpolarizing prepulses. Coexpression of SGK1 significantly attenuated the effect of prepulsing on peak currents. Mutation of Ser to Asp or Ala in the putative phosphorylation consensus sequence in KCNQ4 significantly decreased the sensitivity to SGK1-coexpression. In conclusion, SGK1 regulates current amplitudes and kinetic properties of KCNQ4 channel activity, an effect sensitive to mutations in the SGK1 consensus sequence of the channel.

Regulation of cardiac shal-related potassium channel Kv 4.3 by serum- and glucocorticoid-inducible kinase isoforms in Xenopus oocytes.

The human cardiac transient outward potassium current I(to) is formed by co-assembly of voltage-dependent K(+) channel (Kv 4.3) pore-forming alpha-subunits with differently spliced K channel interacting protein (KChIP) accessory proteins. I(to) is of considerable importance for the normal course of the cardiac ventricular action potential. The present study was performed to determine whether isoforms of the serum- and glucocorticoid-inducible kinase (SGK) family influence Kv 4.3/KChIP2b channel activity in the Xenopus laevis heterologous expression system. Co-expression of SGK1, but not of SGK2 or SGK3, increased Kv 4.3/KChIP2b channel currents. The up-regulation of the current was not due to changes in the activation curve or changes of channel inactivation. The currents in oocytes expressing Kv 4.3 alone were smaller than those in Kv 4.3/KChIP2b expressing oocytes, but were still stimulated by SGK1. The effect of wild-type SGK1 was mimicked by constitutively active SGK1 (SGK1 S422D) but not by an inactive mutant (SGK1 K127N). The current amplitude increase mediated by SGK1 was not dependent on NEDD4.2 or RAB5, nor did it reflect increased cell surface expression. In conclusion, SGK1 stimulates Kv 4.3 potassium channels expressed in Xenopus oocytes by a novel mechanism distinct from the known NEDD4.2-dependent pathway.