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.

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.

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.

Modulation of human Kv1.5 channel kinetics by N-cadherin.

Kv1.5 is expressed in multiple tissues including heart, brain, macrophages, as well as vascular, airway, and intestinal smooth muscle cells. Kv1.5 currents contribute to cardiac repolarization. In cardiac myocytes Kv1.5 colocalizes with N-cadherin. As Kv1.5 expression increases following establishment of cell-cell contacts and N-cadherin influences the activity of other ion channels, we explored whether N-cadherin participates in the regulation of Kv1.5 activity. To this end, we expressed Kv1.5 in Xenopus oocytes with or without additional expression of N-cadherin. Coexpression of N-cadherin was followed by a approximately 2- to 3-fold increase of Kv1.5 induced current. The effect of N-cadherin was not paralleled by significant alterations of Kv1.5 channel abundance within the oocyte cell membrane but resulted primarily from accelerated recovery from inactivation. In conclusion, N-cadherin modifies Kv1.5 channel activity and is thus a novel candidate signaling molecule participating in the regulation of a variety of functions including cardiac action potential and vascular tone.

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).

Functional coassembly of KCNQ4 with KCNE-beta- subunits 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 influences electrical excitability and cell survival. Accordingly, loss of function mutations of the KCNQ4 gene cause hearing loss in humans and functional k.o.-mice show progressive degeneration of outer hair cells (OHCs). However, characteristic electrophysiological features of the native KCNQ4- carried current I(K,n) in OHCs are not recapitulated by expression of KCNQ4 channels in heterologous expression systems. This might suggest modulation of KCNQ4 by interacting KCNE Beta-subunits, which are known to modify the properties of the closely related KCNQ1. The present study explored whether transcripts of the KCNE isoforms could be identified in OHC mRNA and whether the subunits modulate KCNQ4 function. RT-PCR indeed yielded transcripts of all five KCNEs in OHCs. Coexpression of the KCNE- Beta-subunits with human KCNQ4 in the Xenopus laevis oocyte expression system revealed that all KCNEs modulate KCNQ4 voltage dependence, protein stability and ion selectivity of hKCNQ4 in Xenopus oocytes. The deafness-associated Jervell and Lange- Nielsen syndrome (JLNS) mutation KCNE1(D76N) impairs KCNQ4-function whereas the Romano-Ward syndrome (RWS) mutant KCNE1(S74L), which shows normal hearing in patients, does not impair KCNQ4 channel function. In conclusion, KCNEs are presumably coexpressed with KCNQ4 in hair cells from the organ of Corti and might regulate KCNQ4 functional properties, effects that could be important under physiological and pathophysiological conditions.

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.

Differential roles of S6 domain hinges in the gating of KCNQ potassium channels.

Voltage-gated K+ channel activation is proposed to result from simultaneous bending of all S6 segments away from the central axis, enlarging the aperture of the pore sufficiently to permit diffusion of K+ into the water-filled central cavity. The hinge position for the bending motion of each S6 segment is proposed to be a Gly residue and/or a Pro-Val-Pro motif in Kv1-Kv4 channels. The KCNQ1 (Kv7.1) channel has Ala-336 in the Gly-hinge position and Pro-Ala-Gly. Here we show that mutation of Ala-336 to Gly in KCNQ1 increased current amplitude and shifted the voltage dependence of activation to more negative potentials, consistent with facilitation of hinge activity that favors the open state. In contrast, mutation of Ala-336 to Cys or Thr shifted the voltage dependence of activation to more positive potentials and reduced current amplitude. Mutation of the putative Gly hinge to Ala in KCNQ2 (Kv7.2) abolished channel function. Mutation-dependent changes in current amplitude, but not kinetics, were found in heteromeric KCNQ1/KCNE1 channels. Mutation of the Pro or Gly of the Pro-Ala-Gly motif to Ala abolished KCNQ1 function and introduction of Gly in front of the Ala-mutations partially recovered channel function, suggesting that flexibility at the PAG is important for channel activation.

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.