Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate.

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing Asparagine

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehy-drated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMS structure, and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

Voltage-gated proton channels from fungi highlight role of peripheral regions in channel activation

Here, we report the identification and characterization of the first proton channels from fungi. The fungal proteins are related to animal voltage-gated Hv channels and are conserved in both higher and lower fungi. Channels from Basidiomycota and Ascomycota appear to be evolutionally and functionally distinct. Representatives from the two phyla share several features with their animal counterparts, including structural organization and strong proton selectivity, but they differ from each other and from animal Hvs in terms of voltage range of activation, pharmacology, and pH sensitivity. The activation gate of Hv channels is believed to be contained within the transmembrane core of the protein and little is known about contributions of peripheral regions to the activation mechanism. Using a chimeragenesis approach, we find that intra- and extracellular peripheral regions are main determinants of the voltage range of activation in fungal channels, highlighting the role of these overlooked components in channel gating.

Crowding-induced opening of the mechanosensitive Piezo1 channel in silico

Mechanosensitive Piezo1 channels are essential mechanotransduction proteins in eukaryotes. Their curved transmembrane domains, called arms, create a convex membrane deformation, or footprint, which is predicted to flatten in response to increased membrane tension. Here, using a hyperbolic tangent model, we show that, due to the intrinsic bending rigidity of the membrane, the overlap of neighboring Piezo1 footprints produces a flattening of the Piezo1 footprints and arms. Multiple all-atom molecular dynamics simulations of Piezo1 further reveal that this tension-independent flattening is accompanied by gating motions that open an activation gate in the pore. This open state recapitulates experimentally obtained ionic selectivity, unitary conductance, and mutant phenotypes. Tracking ion permeation along the open pore reveals the presence of intracellular and extracellular fenestrations acting as cation-selective sites. Simulations also reveal multiple potential binding sites for phosphatidylinositol 4,5-bisphosphate. We propose that the overlap of Piezo channel footprints may act as a cooperative mechanism to regulate channel activity.

The activation gate controls steady-state inactivation and recovery from inactivation in Shaker

Despite major advances in the structure determination of ion channels, the sequence of molecular rearrangements at negative membrane potentials in voltage-gated potassium channels of the Shaker family remains unknown. Four major composite gating states are documented during the gating process: closed (C), open (O), open-inactivated (OI), and closed-inactivated (CI). Although many steps in the gating cycle have been clarified experimentally, the development of steady-state inactivation at negative membrane potentials and mandatory gating transitions for recovery from inactivation have not been elucidated. In this study, we exploit the biophysical properties of Shaker-IR mutants T449A/V474C and T449A/V476C to evaluate the status of the activation and inactivation gates during steady-state inactivation and upon locking the channel open with intracellular Cd2+. We conclude that at negative membrane potentials, the gating scheme of Shaker channels can be refined in two aspects. First, the most likely pathway for the development of steady-state inactivation is C→O→OI⇌CI. Second, the OI→CI transition is a prerequisite for recovery from inactivation. These findings are in accordance with the widely accepted view that tight coupling is present between the activation and C-type inactivation gates in Shaker and underscore the role of steady-state inactivation and recovery from inactivation as determinants of excitability.

Conformational changes upon gating of KirBac1.1 into an open-activated state revealed by solid-state NMR and functional assays

The conformational changes required for activation and K+ conduction in inward-rectifier K+ (Kir) channels are still debated. These structural changes are brought about by lipid binding. It is unclear how this process relates to fast gating or if the intracellular and extracellular regions of the protein are coupled. Here, we examine the structural details of KirBac1.1 reconstituted into both POPC and an activating lipid mixture of 3:2 POPC:POPG (wt/wt). KirBac1.1 is a prokaryotic Kir channel that shares homology with human Kir channels. We establish that KirBac1.1 is in a constitutively active state in POPC:POPG bilayers through the use of real-time fluorescence quenching assays and Förster resonance energy transfer (FRET) distance measurements. Multidimensional solid-state NMR (SSNMR) spectroscopy experiments reveal two different conformers within the transmembrane regions of the protein in this activating lipid environment, which are distinct from the conformation of the channel in POPC bilayers. The differences between these three distinct channel states highlight conformational changes associated with an open activation gate and suggest a unique allosteric pathway that ties the selectivity filter to the activation gate through interactions between both transmembrane helices, the turret, selectivity filter loop, and the pore helix. We also identify specific residues involved in this conformational exchange that are highly conserved among human Kir channels.