A Dipeptidyl Aminopeptidase–like Protein Remodels Gating Charge Dynamics in Kv4.2 Channels

Dipeptidyl aminopeptidase–like proteins (DPLPs) interact with Kv4 channels and thereby induce a profound remodeling of activation and inactivation gating. DPLPs are constitutive components of the neuronal Kv4 channel complex, and recent observations have suggested the critical functional role of the single transmembrane segment of these proteins (Zagha, E., A. Ozaita, S.Y. Chang, M.S. Nadal, U. Lin, M.J. Saganich, T. McCormack, K.O. Akinsanya, S.Y. Qi, and B. Rudy. 2005. J. Biol. Chem. 280:18853–18861). However, the underlying mechanism of action is unknown. We hypothesized that a unique interaction between the Kv4.2 channel and a DPLP found in brain (DPPX-S) may remodel the channel's voltage-sensing domain. To test this hypothesis, we implemented a robust experimental system to measure Kv4.2 gating currents and study gating charge dynamics in the absence and presence of DPPX-S. The results demonstrated that coexpression of Kv4.2 and DPPX-S causes a −26 mV parallel shift in the gating charge-voltage (Q-V) relationship. This shift is associated with faster outward movements of the gating charge over a broad range of relevant membrane potentials and accelerated gating charge return upon repolarization. In sharp contrast, DPPX-S had no effect on gating charge movements of the Shaker B Kv channel. We propose that DPPX-S destabilizes resting and intermediate states in the voltage-dependent activation pathway, which promotes the outward gating charge movement. The remodeling of gating charge dynamics may involve specific protein–protein interactions of the DPPX-S's transmembrane segment with the voltage-sensing and pore domains of the Kv4.2 channel. This mechanism may determine the characteristic fast operation of neuronal Kv4 channels in the subthreshold range of membrane potentials

Voltage-dependent Gating Rearrangements in the Intracellular T1–T1 Interface of a K+ Channel

The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a “hanging gondola” below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1–S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn2+ site thiolate groups in the T1–T1 interface; therefore, we concluded that the T1–T1 interface is functionally active and not protected by Zn2+ (Wang, G., M. Shahidullah, C.A. Rocha, C. Strang, P.J. Pfaffinger, and M. Covarrubias. 2005. J. Gen. Physiol. 126:55–69). Here, we co-expressed Kv4.1 channels and auxiliary subunits (KChIP-1 and DPPX-S) to investigate the state and voltage dependence of the accessibility of MTSET to the three interfacial cysteines in the T1 domain. The results showed that the average MTSET modification rate constant (kMTSET) is dramatically enhanced in the activated state relative to the resting and inactivated states (∼260- and ∼47-fold, respectively). Crucially, under three separate conditions that produce distinct activation profiles, kMTSET is steeply voltage dependent in a manner that is precisely correlated with the peak conductance–voltage relations. These observations strongly suggest that Kv4 channel gating is tightly coupled to voltage-dependent accessibility changes of native T1 cysteines in the intersubunit Zn2+ site. Furthermore, cross-linking of cysteine pairs across the T1–T1 interface induced substantial inhibition of the channel, which supports the functionally dynamic role of T1 in channel gating. Therefore, we conclude that the complex voltage-dependent gating rearrangements of eukaryotic Kv channels are not limited to the membrane-spanning core but must include the intracellular T1–T1 interface. Oxidative stress in excitable tissues may perturb this interface to modulate Kv4 channel function

A-Type KV Channels in Dorsal Root Ganglion Neurons: Diversity, Function, and Dysfunction

A-type voltage-gated potassium (Kv) channels are major regulators of neuronal excitability that have been mainly characterized in the central nervous system. By contrast, there is a paucity of knowledge about the molecular physiology of these Kv channels in the peripheral nervous system, including highly specialized and heterogenous dorsal root ganglion (DRG) neurons. Although all A-type Kv channels display pore-forming subunits with similar structural properties and fast inactivation, their voltage-, and time-dependent properties and modulation are significantly different. These differences ultimately determine distinct physiological roles of diverse A-type Kv channels, and how their dysfunction might contribute to neurological disorders. The importance of A-type Kv channels in DRG neurons is highlighted by recent studies that have linked their dysfunction to persistent pain sensitization. Here, we review the molecular neurophysiology of A-type Kv channels with an emphasis on those that have been identified and investigated in DRG nociceptors (Kv1.4, Kv3.4, and Kv4s). Also, we discuss evidence implicating these Kv channels in neuropathic pain resulting from injury, and present a perspective of outstanding challenges that must be tackled in order to discover novel treatments for intractable pain disorders

Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation

Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 μM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation. © 2018 Yang et al

Kv4 Channels Underlie the Subthreshold-Operating A-type K+-current in Nociceptive Dorsal Root Ganglion Neurons

The dorsal root ganglion (DRG) contains heterogeneous populations of sensory neurons including primary nociceptive neurons and C-fibers implicated in pain signaling. Recent studies have demonstrated DRG hyperexcitability associated with downregulation of A-type K+ channels; however, the molecular correlate of the corresponding A-type K+ current (IA) has remained hypothetical. Kv4 channels may underlie the IA in DRG neurons. We combined electrophysiology, molecular biology (Whole-Tissue and Single-Cell RT-PCR) and immunohistochemistry to investigate the molecular basis of the IA in acutely dissociated DRG neurons from 7- to 8-day-old rats. Whole-cell recordings demonstrate a robust tetraethylammonium-resistant (20 mM) and 4-aminopyridine-sensitive (5 mM) IA. Matching Kv4 channel properties, activation and inactivation of this IA occur in the subthreshold range of membrane potentials and the rate of recovery from inactivation is rapid and voltage-dependent. Among Kv4 transcripts, the DRG expresses significant levels of Kv4.1 and Kv4.3 mRNAs. Also, single small-medium diameter DRG neurons (∼30 μm) exhibit correlated frequent expression of mRNAs encoding Kv4.1 and Nav1.8, a known nociceptor marker. In contrast, the expressions of Kv1.4 and Kv4.2 mRNAs at the whole-tissue and single-cell levels are relatively low and infrequent. Kv4 protein expression in nociceptive DRG neurons was confirmed by immunohistochemistry, which demonstrates colocalization of Kv4.3 and Nav1.8, and negligible expression of Kv4.2. Furthermore, specific dominant-negative suppression and overexpression strategies confirmed the contribution of Kv4 channels to IA in DRG neurons. Contrasting the expression patterns of Kv4 channels in the central and peripheral nervous systems, we discuss possible functional roles of these channels in primary sensory neurons

Total replacement of recycled aggregate and treated wastewater: concrete recycling in extremis

Million tons of construction and demolition waste (CDW) are generated every year around the world, and most of them are not adequately disposed, generating significant pollution on water, soil and air. Additionally, the use of freshwater in industrial processes, such as the production of cement, concrete manufacturing and curing for newly-built structures; has damaged the health of our freshwater ecosystems, reducing their volume and hindering their natural cycle of renovation. Therefore, the incorporation of recycled aggregate (RA) and treated wastewater (TW) as substitutes for the usual aggregates (UA) and freshwater, could generate significant environmental benefits. In this research, a comparative analysis of the experimental results of the properties of fresh and hardened concrete with different replacement percentage of UA for RA, is presented; and as an innovation the use TW. The results show that, regardless of the replacement percentage and use of treated wastewater, a concrete with RA and TW (recycled concrete in extremis, CRiE) had a satisfactory and acceptable or equivalent performance, not differing significantly from the performance of conventional concrete (CC), confirming that the use of RA for concrete building is feasible.Peer ReviewedPostprint (published version

Gating Charge Immobilization in Kv4.2 Channels: The Basis of Closed-State Inactivation

Kv4 channels mediate the somatodendritic A-type K+ current (ISA) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin (CTX)-sensitive Kv4.2 channel, which enabled us to obtain the first measurements of Kv4.2 gating currents after blocking K+ conduction with CTX (Dougherty and Covarrubias. 2006J. Gen. Physiol. 128:745–753). Here, we exploited this approach further to investigate the mechanism that links closed-state inactivation to slow Q-immobilization in Kv4 channels. The main observations revealed profound Q-immobilization at steady-state over a range of hyperpolarized voltages (−110 to −75 mV). Depolarization in this range moves <5% of the observable Q associated with activation and is insufficient to open the channels significantly. The kinetics and voltage dependence of Q-immobilization and ionic current inactivation between −153 and −47 mV are similar and independent of the channel's proximal N-terminal region (residues 2–40). A coupled state diagram of closed-state inactivation with a quasi-absorbing inactivated state explained the results from ionic and gating current experiments globally. We conclude that Q-immobilization and closed-state inactivation at hyperpolarized voltages are two manifestations of the same process in Kv4.2 channels, and propose that inactivation in the absence of N- and P/C-type mechanisms involves desensitization to voltage resulting from a slow conformational change of the voltage sensors, which renders the channel's main activation gate reluctant to open

Insight into the Modulation of Shaw2 Kv Channels by General Anesthetics: Structural and Functional Studies of S4-S5 linker and S6 C-terminal peptides in micelles by NMR

The modulation of the Drosophila Shaw2 Kv channel by 1-alkanols and inhaled anesthetics is correlated with the involvement of the S4–S5 linker and C-terminus of S6, and consistent with stabilization of the channel\u27s closed state. Structural analysis of peptides from S4–S5 (L45) and S6 (S6c), by nuclear magnetic resonance and circular dichroism spectroscopy supports that an α-helical conformation was adopted by L45, while S6c was only in an unstable/dynamic partially folded α-helix in dodecylphosphocholine micelles. Solvent accessibility and paramagnetic probing of L45 revealed that L45 lies parallel to the surface of micelles with charged and polar residues pointing towards the solution while hydrophobic residues are buried inside the micelles. Chemical shift perturbation introduced by 1-butanol on residues Gln320, Thr321, Phe322 and Arg323 of L45, as well as Thr423 and Gln424 of S6c indicates possible anesthetic binding sites on these two important components in the channel activation apparatus. Diffusion measurements confirmed the association of L45, S6c and 1-butanol with micelles which suggests the capability of 1-butanol to influence a possible interaction of L45 and S6c in the micelle environment