Order and disorder in intermediate filament proteins

AbstractIntermediate filaments (IFs), important components of the cytoskeleton, provide a versatile, tunable network of self-assembled proteins. IF proteins contain three distinct domains: an α-helical structured rod domain, flanked by intrinsically disordered head and tail domains. Recent studies demonstrated the functional importance of the disordered domains, which differ in length and amino-acid sequence among the 70 different human IF genes. Here, we investigate the biophysical properties of the disordered domains, and review recent findings on the interactions between them. Our analysis highlights key components governing IF functional roles in the cytoskeleton, where the intrinsically disordered domains dictate protein–protein interactions, supramolecular assembly, and macro-scale order

KCNE1 Constrains the Voltage Sensor of Kv7.1 K+ Channels

Kv7 potassium channels whose mutations cause cardiovascular and neurological disorders are members of the superfamily of voltage-gated K+ channels, comprising a central pore enclosed by four voltage-sensing domains (VSDs) and sharing a homologous S4 sensor sequence. The Kv7.1 pore-forming subunit can interact with various KCNE auxiliary subunits to form K+ channels with very different gating behaviors. In an attempt to characterize the nature of the promiscuous gating of Kv7.1 channels, we performed a tryptophan-scanning mutagenesis of the S4 sensor and analyzed the mutation-induced perturbations in gating free energy. Perturbing the gating energetics of Kv7.1 bias most of the mutant channels towards the closed state, while fewer mutations stabilize the open state or the inactivated state. In the absence of auxiliary subunits, mutations of specific S4 residues mimic the gating phenotypes produced by co-assembly of Kv7.1 with either KCNE1 or KCNE3. Many S4 perturbations compromise the ability of KCNE1 to properly regulate Kv7.1 channel gating. The tryptophan-induced packing perturbations and cysteine engineering studies in S4 suggest that KCNE1 lodges at the inter-VSD S4-S1 interface between two adjacent subunits, a strategic location to exert its striking action on Kv7.1 gating functions

S1 Constrains S4 in the Voltage Sensor Domain of Kv7.1 K+ Channels

Voltage-gated K+ channels comprise a central pore enclosed by four voltage-sensing domains (VSDs). While movement of the S4 helix is known to couple to channel gate opening and closing, the nature of S4 motion is unclear. Here, we substituted S4 residues of Kv7.1 channels by cysteine and recorded whole-cell mutant channel currents in Xenopus oocytes using the two-electrode voltage-clamp technique. In the closed state, disulfide and metal bridges constrain residue S225 (S4) nearby C136 (S1) within the same VSD. In the open state, two neighboring I227 (S4) are constrained at proximity while residue R228 (S4) is confined close to C136 (S1) of an adjacent VSD. Structural modeling predicts that in the closed to open transition, an axial rotation (∼190°) and outward translation of S4 (∼12 Å) is accompanied by VSD rocking. This large sensor motion changes the intra-VSD S1–S4 interaction to an inter-VSD S1–S4 interaction. These constraints provide a ground for cooperative subunit interactions and suggest a key role of the S1 segment in steering S4 motion during Kv7.1 gating

A Tale of Switched Functions: From Cyclooxygenase Inhibition to M-Channel Modulation in New Diphenylamine Derivatives

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K+ channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K+ channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers

Phosphorylation-Induced Mechanical Regulation of Intrinsically Disordered Neurofilament Proteins

The biological function of protein assemblies has been conventionally equated with a unique three-dimensional protein structure and protein-specific interactions. However, in the past 20 years it has been found that some assemblies contain long flexible regions that adopt multiple structural conformations. These include neurofilament proteins that constitute the stress-responsive supportive network of neurons. Herein, we show that the macroscopic properties of neurofilament networks are tuned by enzymatic regulation of the charge found on the flexible protein regions. The results reveal an enzymatic (phosphorylation) regulation of macroscopic properties such as orientation, stress response, and expansion in flexible protein assemblies. Using a model that explains the attractive electrostatic interactions induced by enzymatically added charges, we demonstrate that phosphorylation regulation is far richer and versatile than previously considered

Gating parameters of WT and mutant Kv7.1 channels.

<p>V₅₀ (half activation voltage) and z (equivalent gating charge) were derived from fitting single Boltzmann function; I₆₀ corresponds to the current density measured at +60 mV in pA/pF. ΔG₀ and ΔΔG₀^c were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001943#s4" target="_blank">methods</a>. Data are expressed as mean ± SEM and in parentheses are indicated the number of cells.^*, p<0.05 compared to WT (two-tailed, Student's unpaired t test). NA, not applicable as R231W mutant is a constitutively open K⁺ leak channel.</p

Mutations stabilizing Kv7.1 to the open state.

<p>(A) and (B) Representative current traces of WT and A226W, respectively. From a holding potential of −90 mV, the membrane was stepped for 3 s from −70 mV to +60 mV in 10 mV increments and then repolarized for 1.5 s to −60 mV to generate the tail currents. (C) and (D) Normalized conductance was plotted as a function of step voltages, for the mutants (black squares) A226W (n = 6) and V241W (n = 11), respectively, and compared to WT (n = 20) (open squares). The activation curves were fitted using one Boltzmann function. (E) Representative current traces of R231W. Membrane was stepped for 3 s from −140 mV to +60 mV in 20 mV increments and then repolarized for 1.5 s to −60 mV. (F) Current-voltage relations of R231W (n = 8) (black squares) and WT (open squares). Current density (pA/pF) was plotted as a function of step voltages.</p

Impact of KCNE1 expression on WT Kv7.1 and mutant R228C.

<p>(A) Representative trace of WT Kv7.1 coexpressed with WT KCNE1. (B) Effects of external Cu-Phen on mutant R228C. Oocytes were bathed in ND96 in the absence and presence of 100 µM Cu-Phen. Shown are representative traces and current-voltage relations were determined as indicated. (C) Shown are representative traces and current-voltage relations of R228C+WT KCNE1 channels, when oocytes were bathed with ND96 in the absence of presence of 100 µM Cu-Phen. Also shown, is the reversal by DTT of the current decrease produced by Cu-Phen. (D) Representative traces of R228C+WT KCNE1 channels, when oocytes were bathed with ND96 containing 100 µM Cu-Phen. Currents were evoked by a train of step depolarization to +30 mV. Similar results have been obtained in 5 other cells.</p