Pablo Miranda

The neuronal-specific SGK1.1 kinase regulates δ-epithelial Na+ channel independently of PY motifs and couples it to phospholipase C signaling

The δ-subunit of the epithelial Na(+) channel (ENaC) is expressed in neurons of the human and monkey central nervous system and forms voltage-independent, amiloride-sensitive Na(+) channels when expressed in heterologous systems. It has been proposed that δ-ENaC could affect neuronal excitability and participate in the transduction of ischemic signals during hypoxia or inflammation. The regulation of δ-ENaC activity is poorly understood. ENaC channels in kidney epithelial cells are regulated by the serum- and glucocorticoid-induced kinase 1 (SGK1). Recently, a new isoform of this kinase (SGK1.1) has been described in the central nervous system. Here we show that δ-ENaC isoforms and SGK1.1 are coexpressed in pyramidal neurons of the human and monkey (Macaca fascicularis) cerebral cortex. Coexpression of δβγ-ENaC and SGK1.1 in Xenopus oocytes increases amiloride-sensitive current and channel plasma membrane abundance. The kinase also exerts its effect when δ-subunits are expressed alone, indicating that the process is not dependent on accessory subunits or the presence of PY motifs in the channel. Furthermore, SGK1.1 action depends on its enzymatic activity and binding to phosphatidylinositol(4,5)-bisphosphate. Physiological or pharmacological activation of phospholipase C abrogates SGK1.1 interaction with the plasma membrane and modulation of δ-ENaC. Our data support a physiological role for SGK1.1 in the regulation of δ-ENaC through a pathway that differs from the classical one and suggest that the kinase could serve as an integrator of different signaling pathways converging on the channel.

Thermodynamic and Kinetic Properties of Amino-Terminal and S4-S5 Loop HERG Channel Mutants under Steady-State Conditions

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K(+) channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of z(g) values and a less negative DeltaG(o), indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased DeltaG(o), indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.

FRET with multiply labeled HERG K+ channels as a reporter of the in vivo coarse architecture of the cytoplasmic domains

The intracellular N-terminus of human ether-a-go-go-related gene (HERG) potassium channels constitutes a key determinant of activation and deactivation characteristics and is necessary for hormone-induced modifications of gating properties. However, the general organization of the long amino and carboxy HERG terminals remains unknown. In this study we performed fluorescence resonance energy transfer (FRET) microscopy with a library of fluorescent HERG fusion proteins obtained combining site-directed and transposon-based random insertion of GFP variants into multiple sites of HERG. Determinations of FRET efficiencies with functional HERG channels labeled in different combinations localize the fluorophores, introduced in the amino and carboxy ends, in two quadratic planes of 7.8 and 8.6 nm lateral size, showing a vertical separation of nearly 8 nm without major angular torsion between the planes. Similar analysis using labels at positions 345 and 905 of the amino and carboxy terminals, located them slightly above the planes delimited by the amino and carboxy end labels, respectively. Our data also indicate an almost vertical arrangement of the fluorophores introduced in the NH(2) and COOH ends and at position 905, but a near 45 degrees angular rotation between the planes delimited by these labels and the 345-located fluorophores. Systematic triangulation using interfluorophore distances coming from multiply labeled channels provides an initial constraint on the overall in vivo arrangement of the HERG cytoplasmic domains, suggesting that the C-linker/CNBD region of HERG hangs centrally below the transmembrane core, with the initial portion of the amino terminus around its top and side surfaces directed towards the gating machinery.

Relevance of the proximal domain in the amino‐terminus of HERG channels for regulation by a phospholipase C‐coupled hormone receptor

We used Xenopus oocytes co‐expressing thyrotropin‐releasing hormone (TRH) receptors and human ether‐a‐go‐go‐related gene (HERG) K+ channel variants carrying different amino‐terminal modifications to check the relevance of the proximal domain for hormonal regulation of the channel. Deletion of the whole proximal domain (Δ138–373) eliminates TRH‐induced modifications in activation and deactivation parameters. TRH effects on activation are also suppressed with channels lacking the second half of the proximal domain or only residues 326–373. However, normal responses to TRH are obtained with Δ346–373 channels. Thus, whereas residues 326–345 are required for the hormonal modulation of HERG activation, different proximal domain sequences contribute to set HERG gating characteristics and its regulation by TRH.

Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane

The epithelial Na(+) channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na(+) reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, α, β, and γ. A fourth ENaC subunit, named δ, is expressed in the nervous system of primates, where its role is unknown. The human δ-ENaC gene generates at least two splice isoforms, δ(1) and δ(2) , differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either δ(1) or δ(2) , with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of δ(1) in Xenopus oocytes and HEK293 cells produces higher current levels than δ(2) . Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of β- and γ-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in δ(2)-N terminus. Analysis of δ(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The δ(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that δ-ENaC undergoes dynamin-independent endocytosis as opposed to αβγ-channels.

Specificity of TRH receptor coupling to G‐proteins for regulation of ERG K+ channels in GH3 rat anterior pituitary cells

The identity of the G‐protein coupling thyrotropin‐releasing hormone (TRH) receptors to rat ether‐à‐go‐go related gene (r‐ERG) K+ channel modulation was studied in situ using perforated‐patch clamped adenohypophysial GH3 cells and dominant‐negative variants (Gα‐QL/DN) of G‐protein α subunits. Expression of dominant‐negative Gαq/11 that minimizes the TRH‐induced Ca2+ signal had no effect on r‐ERG current inhibition elicited by the hormone. In contrast, the introduction of dominant‐negative variants of Gα13 and the small G‐protein Rho caused a significant loss of the inhibitory effect of TRH on r‐ERG. A strong reduction of this TRH effect was also obtained in cells expressing either dominant‐negative Gαs or transducin α subunits, an agent known to sequester free G‐protein βγ dimers. As a further indication of specificity of the dominant‐negative effects, only the dominant‐negative variants of Gα13 and Rho (but not Gαs‐QL/DN or Gαt) were able to reduce the TRH‐induced shifts of human ERG (HERG) activation voltage dependence in HEK293 cells permanently expressing HERG channels and TRH receptors. Our results demonstrate that whereas the TRH receptor uses a Gq/11 protein for transducing the Ca2+ signal during the initial response to TRH, this G‐protein is not involved in the TRH‐induced inhibition of endogenous r‐ERG currents in pituitary cells. They also identify Gs (or a Gs‐like protein) and G13 as important contributors to the hormonal effect in these cells and suggest that βγ dimers released from these proteins may participate in modulation of ERG currents triggered by TRH.

Role of BK Potassium Channels Shaping Action Potentials and the Associated [Ca2+]i Oscillations in GH3 Rat Anterior Pituitary Cells

Measurements of electrical activity and intracellular Ca2+ levels were performed in perforated-patch clamped GH3 cells to determine the contribution of large-conductance calcium-activated K+ (BK) channels to action potential repolarization and size of the associated Ca2+ oscillations. By examining the dependence of action potential (AP) duration on extracellular Ca2+ levels in the presence and the absence of the specific BK channel blocker paxilline, it is observed that plateau-like action potentials are associated to low densities of paxilline-sensitive currents. Extracellular Ca2+ increases or paxilline additions are not able to largely modify action potential duration in cells showing a reduced expression of BK currents. Furthermore, specific blockade of these currents with paxilline systematically elongates AP duration, but only under conditions in which short APs and/or prominent BK currents recorded under voltage-clamp mode are present in the same cells. Our data indicate that in GH3 cells, BK channels act primarily ending the action potential and suggest that by contributing to fine-tuning cellular electrical properties and hence intracellular Ca2+ variations, BK channels may play an important role on time- and cell-dependent modulation of physiological outputs in adenohypophyseal cells.

Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A – helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.

The Neuronal Serum- and Glucocorticoid-Regulated Kinase 1.1 Reduces Neuronal Excitability and Protects against Seizures through Upregulation of the M-Current

The M-current formed by tetramerization of Kv7.2 and Kv7.3 subunits is a neuronal voltage-gated K+ conductance that controls resting membrane potential and cell excitability. In Xenopus laevis oocytes, an increase in Kv7.2/3 function by the serum- and glucocorticoid-regulated kinase 1 (SGK1) has been reported previously (Schuetz et al., 2008). We now show that the neuronal isoform of this kinase (SGK1.1), with distinct subcellular localization and modulation, upregulates the Kv7.2/3 current in Xenopus oocytes and mammalian human embryonic kidney HEK293 cells. In contrast to the ubiquitously expressed SGK1, the neuronal isoform SGK1.1 interacts with phosphoinositide-phosphatidylinositol 4,5-bisphosphate (PIP2) and is distinctly localized to the plasma membrane (Arteaga et al., 2008). An SGK1.1 mutant with disrupted PIP2 binding sites produced no effect on Kv7.2/3 current amplitude. SGK1.1 failed to modify the voltage dependence of activation and did not change activation or deactivation kinetics of Kv7.2/3 channels. These results suggest that the kinase increases channel membrane abundance, which was confirmed with flow cytometry assays. To evaluate the effect of the kinase in neuronal excitability, we generated a transgenic mouse (Tg.sgk) expressing a constitutively active form of SGK1.1 (S515D). Superior cervical ganglion (SCG) neurons isolated from Tg.sgk mice showed a significant increase in M-current levels, paralleled by reduced excitability and more negative resting potentials. SGK1.1 effect on M-current in Tg.sgk–SCG neurons was counteracted by muscarinic receptor activation. Transgenic mice with increased SGK1.1 activity also showed diminished sensitivity to kainic acid-induced seizures. Altogether, our results unveil a novel role of SGK1.1 as a physiological regulator of the M-current and neuronal excitability.

Participation of HERG channel cytoplasmic structures on regulation by the G protein-coupled TRH receptor

Human ether-a-go-go-related gene (HERG) channels heterologously expressed in Xenopus oocytes are regulated by the activation of G protein-coupled hormone receptors that, like the thyrotropin-releasing hormone (TRH) receptor, activate phospholipase C. Previous work with serially deleted HERG mutants suggested that residues 326–345 located in the proximal domain of the channels amino terminus might be required for the hormonal modulation of HERG activation. Generation of new channel mutants deleted in this region further point to the amino acid sequence between residues 326 and 332 as a possible determinant of the TRH effects, but individual or combined single-point mutations in this sequence demonstrate that maintenance of its consensus sites for phosphorylation and/or interaction with regulatory components is not important for the modulatory response(s). The TRH-induced effects also remained unaltered when a basic amino acid cluster located between residues 362 and 366 is eliminated. Additionally, no effect of TRH was observed in channels carrying single-point mutations at the beginning of the intracellular loop linking transmembrane domains S4 and S5. Our results indicate that a correct structural arrangement of the amino terminal domains is essential for the hormone-induced modifications of HERG activation. They also suggest that the hormonal regulatory action is transmitted to the transmembrane channel core through interactions between the cytoplasmic domains and the initial portion of the S4–S5 linker.