A unique mechanism of inactivation gating of the Kv channel family member Kv7.1 and its modulation by PIP2 and calmodulin.

Inactivation of voltage-gated K+ (Kv) channels mostly occurs by fast N-type or/and slow C-type mechanisms. Here, we characterized a unique mechanism of inactivation gating comprising two inactivation states in a member of the Kv channel superfamily, Kv7.1. Removal of external Ca2+ in wild-type Kv7.1 channels produced a large, voltage-dependent inactivation, which differed from N- or C-type mechanisms. Glu295 and Asp317 located, respectively, in the turret and pore entrance are involved in Ca2+ coordination, allowing Asp317 to form H-bonding with the pore helix Trp304, which stabilizes the selectivity filter and prevents inactivation. Phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+-calmodulin prevented Kv7.1 inactivation triggered by Ca2+-free external solutions, where Ser182 at the S2-S3 linker relays the calmodulin signal from its inner boundary to the external pore to allow proper channel conduction. Thus, we revealed a unique mechanism of inactivation gating in Kv7.1, exquisitely controlled by external Ca2+ and allosterically coupled by internal PIP2 and Ca2+-calmodulin.

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions.

Inactivation is an intrinsic property of numerous voltage-gated K+ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.

Ca2+-Calmodulin and PIP2 interactions at the proximal C-terminus of Kv7 channels.

In the heart, co-assembly of Kv7.1 with KCNE1 produces the slow IKS potassium current, which repolarizes the cardiac action potential and mutations in human Kv7.1 and KCNE1 genes cause cardiac arrhythmias. The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2) and recently we revealed the competition of PIP2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation. Data indicated that PIP2 and Ca2+-CaM perform the same function on IKS channel gating to stabilize the channel open state. Here we show that similar features were observed for Kv7.1 currents expressed alone. We also find that conservation of homologous residues in helix B of other Kv7 subtypes confer similar competition of Ca2+-CaM with PIP2 binding to their proximal C-termini and suggest that PIP2-CaM interactions converge to Kv7 helix B to modulates channel activity in a Kv7 subtype-dependent manner.

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition.

Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases.

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

Structural Insights into the M-Channel Proximal C-Terminus/Calmodulin Complex.

The Kv7 (KCNQ) channel family, comprising voltage-gated potassium channels, plays major roles in fine-tuning cellular excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminal (CT) domain bound constitutively by calmodulin (CaM). This domain plays key functions in channel tetramerization, trafficking, and gating. CaM binds to the proximal CT, comprising helices A and B. Kv7.2 and Kv7.3 are expressed in neural tissues. Together, they form the heterotetrameric M channel. We characterized Kv7.2, Kv7.3, and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by solution methods at various Ca(2+)concentrations and determined them all to have a 1:1 stoichiometry. We then determined the crystal structure of the Q3A-Q2B/CaM complex at high Ca(2+) concentration to 2.0 Å resolution. CaM hugs the antiparallel coiled coil of helices A and B, braced together by an additional helix. The structure displays a hybrid apo-Ca(2+) CaM conformation even though four Ca(2+) ions are bound. Our results pinpoint unique interactions enabling the possible intersubunit pairing of Kv7.3 helix A and Kv7.2 helix B while underlining the potential importance of Kv7.3 helix A's role in stabilizing channel oligomerization. Also, the structure can be used to rationalize various channelopathic mutants. Functional testing of the chimeric channel found it to have a voltage-dependence similar to the M channel, thereby demonstrating helix A's importance in imparting gating properties.

SOCS1 in cancer: An oncogene and a tumor suppressor.

The Suppressor Of Cytokine Signaling 1 (SOCS1) has been extensively investigated in immune cells where it works as a potent inhibitor of inflammation by negative feedback regulation of the cytokine-activated JAK-STAT signaling pathways. SOCS1 is also recognized as a tumor suppressor in numerous cancers and its critical functional relevance in non-immune cells, including epithelial cells, has just begun to emerge. Most notably, conflicting results from clinical and experimental studies suggest that SOCS1 may function as either a tumor suppressor or a tumor promoter, in a cell context-dependent manner. Here, we present an overview of the mechanisms underlying SOCS1 function as a tumor suppressor and discuss the emerging evidences of SOCS1 activity as an oncogene.

Tumour-promoting role of SOCS1 in colorectal cancer cells.

The SOCS1 (Suppressor Of Cytokine Signalling 1) protein is considered a tumour suppressor. Notably, the SOCS1 gene is frequently silenced in cancer by hypermethylation of its promoter. Besides blocking inflammation, SOCS1 tumour suppressor activity involves Met receptor inhibition and enhancement of p53 tumour suppressor activity. However, the role of SOCS1 in colorectal cancer (CRC) remains understudied and controversial. Here, we investigated SOCS1 relevance for CRC by querying gene expression datasets of human CRC specimens from The Cancer Genome Atlas (TCGA), and by SOCS1 gain/loss-of-function analyses in murine and human colon carcinoma cells. Our results show that SOCS1 mRNA levels in tumours were more often elevated than reduced with respect to matched adjacent normal tissue of CRC specimens (n = 41). The analysis of TCGA dataset of 431 CRC patients revealed no correlation between SOCS1 expression and overall survival. Overexpression of SOCS1 in CRC cells triggered cell growth enhancement, anchorage-independent growth and resistance to death stimuli, whereas knockdown of SOCS1 reduced these oncogenic features. Moreover, SOCS1 overexpression in mouse CT26 cells increased tumourigenesis in vivo. Biochemical analyses showed that SOCS1 pro-oncogenic activity correlated with the down-modulation of STAT1 expression. Collectively, these results suggest that SOCS1 may work as an oncogene in CRC.

Structural basis of a Kv7.1 potassium channel gating module: studies of the intracellular c-terminal domain in complex with calmodulin.

Kv7 channels tune neuronal and cardiomyocyte excitability. In addition to the channel membrane domain, they also have a unique intracellular C-terminal (CT) domain, bound constitutively to calmodulin (CaM). This CT domain regulates gating and tetramerization. We investigated the structure of the membrane proximal CT module in complex with CaM by X-ray crystallography. The results show how the CaM intimately hugs a two-helical bundle, explaining many channelopathic mutations. Structure-based mutagenesis of this module in the context of concatemeric tetramer channels and functional analysis along with in vitro data lead us to propose that one CaM binds to one individual protomer, without crosslinking subunits and that this configuration is required for proper channel expression and function. Molecular modeling of the CT/CaM complex in conjunction with small-angle X-ray scattering suggests that the membrane proximal region, having a rigid lever arm, is a critical gating regulator.