Slow Inactivation Process Research Articles

Analysis of the role of Na<sub>v</sub>1.5 slow inactivation in the development of inherited cardiac pathology

Voltage-gated cardiac sodium channels Nav1.5 are responsible for the initiation and propagation of action potentials in cardiomyocytes. Dysfunction of Nav1.5 can be caused both by pathogenic variants in the SCN5A gene itself, which encodes Nav1.5, and by genetic variants in the genes of other proteins, regulating channel activity and trafficking. The change of different phases of the action potential is determined by the strict temporal organization of activation and inactivation of various ion channels. Transitions between channel functional states (for example, to slow inactivated state) can be influenced by various factors and proteins interacting with the channel. Despite the fact that the process of slow inactivation of the channel has been known for several decades, its role in the mechanism of development of hereditary heart pathology remains unclear. In this work, using the patch clamp method in whole-cell leads, we studied changes in the process of slow Nav1.5 inactivation under the influence of various mutations in structural genes (DSP-H1684R, LMNA-R249Q, FLNC-R1267Q, FLNC-V2264M) associated with a genetically determined myocardial pathology leading to dysfunction of cardiomyocytes. The study used a model of cardiomyocytes differentiated from induced pluripotent stem cells (СM-iPSCs). We have demonstrated an increase in slow inactivation in the model of CM-iPSCs obtained from patients with a phenotype of cardiomyopathy combined with ventricular arrhythmias. Thus, this work contributes to understanding the role of the slow inactivation process in the mechanism of the development of heart pathology.

Dysfunctional sodium channel kinetics as a novel epilepsy mechanism in chromosome 15q11-q13 duplication syndrome.

Duplication of the maternal chromosome 15q11.2-q13.1 region causes Dup15q syndrome, a highly penetrant neurodevelopmental disorder characterized by severe autism and refractory seizures. Although UBE3A, the gene encoding the ubiquitin ligase E3A, is thought to be the main driver of disease phenotypes, the cellular and molecular mechanisms that contribute to the development of the syndrome are yet to be determined. We previously established the necessity of UBE3A overexpression for the development of cellular phenotypes in human Dup15q neurons, including increased action potential firing and increased inward current density, which prompted us to further investigate sodium channel kinetics. We used a Dup15q patient-derived induced pluripotent stem cell line that was CRISPR-edited to remove the supernumerary chromosome and create an isogenic control line. We performed whole cell patch clamp electrophysiology on Dup15q and corrected control neurons at two time points of in vitro development. Compared to corrected neurons, Dup15q neurons showed increased sodium current density and a depolarizing shift in steady-state inactivation. Moreover, onset of slow inactivation was delayed, and a faster recovery from both fast and slow inactivation processes was observed in Dup15q neurons. A fraction of sodium current in Dup15q neurons (~15%) appeared to be resistant to slow inactivation. Not unexpectedly, a higher fraction of persistent sodium current was also observed in Dup15q neurons. These phenotypes were modulated by the anticonvulsant drug rufinamide. Sodium channels play a crucial role in the generation of action potentials, and sodium channelopathies have been uncovered in multiple forms of epilepsy. For the first time, our work identifies in Dup15q neurons dysfunctional inactivation kinetics, which have been previously linked to multiple forms of epilepsy. Our work can also guide therapeutic approaches to epileptic seizures in Dup15q patients and emphasize the role of drugs that modulate inactivation kinetics, such as rufinamide.

A point mutation turns an inactivated potassium channel into a hyperpolarization activated cation channel.

In potassium channels, the slow inactivation (C-type) process decreases the ionic conduction by affecting the selectivity filter. The W434F mutation in Shaker potassium channel stabilizes it in the inactivated conformation. The recent determination of the structure of the Shaker W434F channel shows a dilated selectivity filter, compared to Shaker WT structure, reminiscent to the hyperpolarization activated cyclic nucleotide gated channel (HCN). This suggests a common link between inactivation and hyperpolarization gating, which is explored in this work. In Shaker the P475D (Sh-P475D) is a mutation in the bundle crossing region that stabilizes the activation gate in the open conformation over a wide voltage range (−140 mV to +100). Sh-P475D was expressed in Xenopus oocytes and inactivation was favored by removing external potassium. Under these conditions, the channel displays robust hyperpolarization activated currents reminiscent of HCN. (Similar currents are observed when the W434F mutation is added in Sh-P475D background.) The currents elicited by hyperpolarizing voltages in the Sh-P475D mutant are non-selective (PK/PNa∼1) and even N-methyl-D-glucamine and calcium are permeable in this mutant. We analyzed the role of residues known to affect pore inactivation, the contribution of the selectivity filter, and the influence of the voltage sensor movement on the kinetics and steady state characteristics of the P475D-induced hyperpolarization activated currents. Our results suggest a mechanism of hyperpolarization gating and inactivation at the selectivity filter that seems to be common among voltage gated cation channels. Supported by NIH GM03076.

Slow Inactivation of Sodium Channels Contributes to Short-Term Adaptation in Vomeronasal Sensory Neurons.

Adaptation plays an important role in sensory systems as it dynamically modifies sensitivity to allow the detection of stimulus changes. The vomeronasal system controls many social behaviors in most mammals by detecting pheromones released by conspecifics. Stimuli activate a transduction cascade in vomeronasal neurons that leads to spiking activity. Whether and how these neurons adapt to stimuli is still debated and largely unknown. Here, we measured short-term adaptation performing current-clamp whole-cell recordings by using diluted urine as a stimulus, as it contains many pheromones. We measured spike frequency adaptation in response to repeated identical stimuli of 2–10 s duration that was dependent on the time interval between stimuli. Responses to paired current steps, bypassing the signal transduction cascade, also showed spike frequency adaptation. We found that voltage-gated Na+ channels in VSNs undergo slow inactivation processes. Furthermore, recovery from slow inactivation of voltage-gated Na+ channels occurs in several seconds, a time scale similar to that measured during paired-pulse adaptation protocols, suggesting that it partially contributes to short-term spike frequency adaptation. We conclude that vomeronasal neurons do exhibit a time-dependent short-term spike frequency adaptation to repeated natural stimuli and that slow inactivation of Na+ channels contributes to this form of adaptation. These findings not only increase our knowledge about adaptation in the vomeronasal system, but also raise the question of whether slow inactivation of Na+ channels may play a role in other sensory systems.

P1597Two novel mutations in SCN5A gene cause enhanced inactivation and lead to Brugada syndrome

Abstract Background Mutations in gene SCN5A, encoding cardiac potential-dependent sodium channel Nav1.5, are associated with various arrhythmogenic disorders among which the Brugada syndrome (BrS) and the Long QT syndrome (LQT) are the best characterized. BrS1 is associated with sodium channel dysfunction, which can be reflected by decreased current, impaired activation and enhanced inactivation. We found two novel mutations in our patients with BrS and explored their effect on fast and slow inactivation of cardiac sodium channel. Purpose The aim of this study was to investigate the effect of BrS (Y739D, L1582P) mutations on different inactivation processes in in vitro model. Methods Y739D and L1582P substitutions were introduced in SCN5A cDNA using site-directed mutagenesis. Sodium currents were recorded at room temperature in transfected HEK293-T cells using patch-clamp technique with holding potential −100 mV. In order to access the fast steady-state inactivation curve we used double-pulse protocol with 10 ms prepulses. To analyze voltage-dependence of slow inactivation we used two-pulse protocol with 10s prepulse, 20ms test pulse and 25ms interpulse at −100mV to allow recovery from fast inactivation. Electrophysiological measurements are presented as mean ±SEM. Results Y739D mutation affects highly conserved tyrosine 739 among voltage-gated sodium and calcium channels in the segment IIS2. Mutation L1582P located in the loop IVS4-S5, and leucine in this position is not conserved among voltage-gated channels superfamily. We have shown that Y739D leads to significant changes in both fast and slow inactivation, whereas L1582P enhanced slow inactivation only. Steady-state fast inactivation for Y739D was shifted on 8.9 mV towards more negative potentials compare with that for WT, while L1582P did not enhanced fast inactivation (V1/2 WT: −62.8±1.7 mV; Y739D: −71.7±2.3 mV; L1582P: −58.7±1.4 mV). Slow inactivation was increased for both substitutions (INa (+20mV)/INa (−100mV) WT: 0.45±0.03; Y739D: 0,34±0.09: L1582P: 0.38±0.04). Steady-state fast inactivation Conclusions Both mutations, observed in patients with Brugada syndrome, influence on the slow inactivation process. Enhanced fast inactivation was shown only for Y739D mutant. The more dramatic alterations in sodium channel biophysical characteristics are likely linked with mutated residue conservativity. Acknowledgement/Funding RSF #17-15-01292

Effects of eslicarbazepine on slow inactivation processes of sodium channels in dentate gyrus granule cells.

Pharmacoresistance is a problem affecting ∼30% of chronic epilepsy patients. An understanding of the mechanisms of pharmacoresistance requires a precise understanding of how antiepileptic drugs interact with their targets in control and epileptic tissue. Although the effects of (S)-licarbazepine (S-Lic) on sodium channel fast inactivation are well understood and have revealed maintained activity in epileptic tissue, it is not known how slow inactivation processes are affected by S-Lic in epilepsy. We have used voltage clamp recordings in isolated dentate granule cells (DGCs) and cortical pyramidal neurons of control versus chronically epileptic rats (pilocarpine model of epilepsy) and in DGCs isolated from hippocampal specimens from temporal lobe epilepsy patients to examine S-Lic effects on sodium channel slow inactivation. S-Lic effects on entry into and recovery from slow inactivation were negligible, even at high concentrations of S-Lic (300 μmol/L). Much more pronounced S-Lic effects were observed on the voltage dependence of slow inactivation, with significant effects at 100 μmol/L S-Lic in DGCs from control and epileptic rats or temporal lobe epilepsy patients. For none of these effects of S-Lic could we observe significant differences either between sham-control and epileptic rats, or between human DGCs and the two animal groups. S-Lic was similarly effective in cortical pyramidal neurons from sham-control and epileptic rats. Finally, we show in expression systems that S-Lic effects on slow inactivation voltage dependence are only observed in Nav 1.2 and Nav 1.6 subunits, but not in Nav 1.1 and Nav 1.3 subunits. From these data, we conclude that a major mechanism of action of S-Lic is an effect on slow inactivation, primarily through effects on slow inactivation voltage dependence of Nav 1.2 and Nav 1.6 channels. Second, we demonstrate that this main effect of S-Lic is maintained in both experimental and human epilepsy and applies to principal neurons of different brain areas.

Activity of the anticonvulsant lacosamide in experimental and human epilepsy via selective effects on slow Na+ channel inactivation.

In human epilepsy, pharmacoresistance to antiepileptic drug therapy is a major problem affecting ~30% of patients with epilepsy. Many classical antiepileptic drugs target voltage-gated sodium channels, and their potent activity in inhibiting high-frequency firing has been attributed to their strong use-dependent blocking action. In chronic epilepsy, a loss of use-dependent block has emerged as a potential cellular mechanism of pharmacoresistance for anticonvulsants acting on voltage-gated sodium channels. The anticonvulsant drug lacosamide (LCM) also targets sodium channels, but has been shown to preferentially affect sodium channel slow inactivation processes, in contrast to most other anticonvulsants. We used whole-cell voltage clamp recordings in acutely isolated cells to investigate the effects of LCM on transient Na+ currents. Furthermore, we used whole-cell current clamp recordings to assess effects on repetitive action potential firing in hippocampal slices. We show here that LCM exerts its effects primarily via shifting the slow inactivation voltage dependence to more hyperpolarized potentials in hippocampal dentate granule cells from control and epileptic rats, and from patients with epilepsy. It is important to note that this activity of LCM was maintained in chronic experimental and human epilepsy. Furthermore, we demonstrate that the efficacy of LCM in inhibiting high-frequency firing is undiminished in chronic experimental and human epilepsy. Taken together, these results show that LCM exhibits maintained efficacy in chronic epilepsy, in contrast to conventional use-dependent sodium channel blockers such as carbamazepine. They also establish that targeting slow inactivation may be a promising strategy for overcoming target mechanisms of pharmacoresistance.

Effect of phenytoin on sodium conductances in rat hippocampal CA1 pyramidal neurons.

The antiepileptic drug phenytoin (PHT) is thought to reduce the excitability of neural tissue by stabilizing sodium channels (NaV) in inactivated states. It has been suggested the fast-inactivated state (IF) is the main target, although slow inactivation (IS) has also been implicated. Other studies on local anesthetics with similar effects on sodium channels have implicated the NaV voltage sensor interactions. In this study, we reexamined the effect of PHT in both equilibrium and dynamic transitions between fast and slower forms of inactivation in rat hippocampal CA1 pyramidal neurons. The effects of PHT were observed on fast and slow inactivation processes, as well as on another identified "intermediate" inactivation process. The effect of enzymatic removal of IF was also studied, as well as effects on the residual persistent sodium current (INaP). A computational model based on a gating charge interaction was derived that reproduced a range of PHT effects on NaV equilibrium and state transitions. No effect of PHT on IF was observed; rather, PHT appeared to facilitate the occupancy of other closed states, either through enhancement of slow inactivation or through formation of analogous drug-bound states. The overall significance of these observations is that our data are inconsistent with the commonly held view that the archetypal NaV channel inhibitor PHT stabilizes fast inactivation states, and we demonstrate that conventional slow activation "IS" and the more recently identified intermediate-duration inactivation process "II" are the primary functional targets of PHT. In addition, we show that the traditional explanatory frameworks based on the "modulated receptor hypothesis" can be substituted by simple, physiologically plausible interactions with voltage sensors. Additionally, INaP was not preferentially inhibited compared with peak INa at short latencies (50 ms) by PHT.

Structural model of the open-closed-inactivated cycle of prokaryotic voltage-gated sodium channels.

In excitable cells, the initiation of the action potential results from the opening of voltage-gated sodium channels. These channels undergo a series of conformational changes between open, closed, and inactivated states. Many models have been proposed for the structural transitions that result in these different functional states. Here, we compare the crystal structures of prokaryotic sodium channels captured in the different conformational forms and use them as the basis for examining molecular models for the activation, slow inactivation, and recovery processes. We compare structural similarities and differences in the pore domains, specifically in the transmembrane helices, the constrictions within the pore cavity, the activation gate at the cytoplasmic end of the last transmembrane helix, the C-terminal domain, and the selectivity filter. We discuss the observed differences in the context of previous models for opening, closing, and inactivation, and present a new structure-based model for the functional transitions. Our proposed prokaryotic channel activation mechanism is then compared with the activation transition in eukaryotic sodium channels.

Inactivation of faecal indicator bacteria in a roof-captured rainwater system under ambient meteorological conditions

In this study, faecal indicator bacteria (FIB) namely Escherichia coli and Enterococcus spp. were seeded into slurries of possum faeces and placed on the roof and in the gutter of a roof-captured rainwater (RCR) system. The persistence of FIB in these circumstances was determined under ambient climatic conditions. FIB persistence was also determined under in situ conditions in tank water using diffusion chambers. The numbers of surviving FIB at different time intervals were enumerated using culture-based methods. Both FIB were rapidly inactivated on the roof under sunlight conditions (T(90) = 2 h) compared with shade conditions (T(90) = 9-53 h). Significant differences were observed between sunlight and shade conditions on the roof for both T90 values of E. coli (P < 0·001) and Enterococcus spp. (P < 0·001). E. coli showed biphasic inactivation patterns under both clean and unclean gutter conditions. Enterococcus spp., however, showed rapid inactivation (T(90) = 2 h for the clean gutter and T(90) = 6 h for the unclean gutter) compared with E. coli (T(90) = 22 h for the clean gutter and T(90) = 20 h for the unclean gutter). Significant differences were also observed between the T(90) values of E. coli and Enterococcus spp. for both clean (P < 0·001) and unclean (P < 0·001) gutters. Both E. coli and Enterococcus spp. showed nonlinear biphasic inactivation in tank water. Significant difference was observed between the T(90) value of E. coli inactivation compared with Enterococcus spp. (P < 0·001) in the tank water. In this study, FIB were observed to survive longer (T(90) = 9-53 h) on the roof under shade conditions compared with sunlight conditions (T(90) = 2 h). If there is a rainfall event within two to three days after the deposition of faecal maters on the roof, it is highly likely that FIB would be transported to the tank water. When introduced into the tank, a relatively slow inactivation process may take place (T(90) = 38-72 h). The presence of FIB in water indicates faecal pollution and potential presence of enteric pathogens. Therefore, the information on the resilience of FIB, as obtained in this study, can be used for indirect assessment of health risks associated with using roof-captured rainwater for potable and nonpotable purposes.

(Biphenyl-4-yl)methylammonium Chlorides: Potent Anticonvulsants That Modulate Na+ Currents

We have reported that compounds containing a biaryl linked unit (Ar-X-Ar') modulated Na(+) currents by promoting slow inactivation and fast inactivation processes and by inducing frequency (use)-dependent inhibition of Na(+) currents. These electrophysiological properties have been associated with the mode of action of several antiepileptic drugs. In this study, we demonstrate that the readily accessible (biphenyl-4-yl)methylammonium chlorides (compound class B) exhibited a broad range of anticonvulsant activities in animal models, and in the maximal electroshock seizure test the activity of (3'-trifluoromethoxybiphenyl-4-yl)methylammonium chloride (8) exceeded that of phenobarbital and phenytoin upon oral administration to rats. Electrophysiological studies of 8 using mouse catecholamine A-differentiated cells and rat embryonic cortical neurons confirmed that 8 promoted slow and fast inactivation in both cell types but did not affect the frequency (use)-dependent block of Na(+) currents.

Discovery of Lacosamide Affinity Bait Agents That Exhibit Potent Voltage-Gated Sodium Channel Blocking Properties

Lacosamide ((R)-1) is a recently marketed, first-in-class, antiepileptic drug. Patch-clamp electrophysiology studies are consistent with the notion that (R)-1 modulates voltage-gated Na(+) channel function by increasing and stabilizing the slow inactivation state without affecting fast inactivation. The molecular pathway(s) that regulate slow inactivation are poorly understood. Affinity baits are chemical reactive units, which when appended to a ligand (drug) can lead to irreversible, covalent modification of the receptor thus permitting drug binding site identification including, possibly, the site of ligand function. We describe, herein, the synthesis of four (R)-1 affinity baits, (R)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-8), (S)-N-(4″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((S)-8), (R)-N-(3″-isothiocyanatobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-9), and (R)-N-(3″-acrylamidobiphenyl-4'-yl)methyl 2-acetamido-3-methoxypropionamide ((R)-10). The affinity bait compounds were designed to interact with the receptor(s) responsible for (R)-1-mediated slow inactivation. We show that (R)-8 and (R)-9 are potent inhibitors of Na(+) channel function and function by a pathway similar to that observed for (R)-1. We further demonstrate that (R)-8 function is stereospecific. The calculated IC50 values determined for Na(+) channel slow inactivation for (R)-1, (R)-8, and (R)-9 were 85.1, 0.1, and 0.2 μM, respectively. Incubating (R)-9 with the neuronal-like CAD cells led to appreciable levels of Na(+) channel slow inactivation after cellular wash, and the level of slow inactivation only modestly decreased with further incubation and washing. Collectively, these findings have identified a promising structural template to investigate the voltage-gated Na(+) channel slow inactivation process.

A Key Gating Charge Interaction Required for Slow Inactivation of the Navab Bacterial Sodium Channel

Bacterial voltage-gated sodium (Nav) channels are considered an ideal model for structure-function studies. The elucidation of the crystal structure of the bacterial channel NavAb (Payandeh et al., Nature 486, 135-139, 2011) opened an avenue to understand electrical signaling in excitable cells at the structural level. NavAb expressed in Hi5 insect cells as for structural studies has unusually negative voltage-dependent activation (Va ∼ 130 mV). NavAb also has three phases of inactivation, a biphasic early inactivation process (τ1∼103 ms, τ2∼3.8 s, at −180 mV) followed by an unusually strong and use-dependent slow-inactivation process. To search for the molecular basis for negative activation and slow inactivation of NavAb, we mutated the outermost gating charge partner in the S2 segment, Asn49, to Lys. This mutation shifted the activation curve ∼ 75 mV toward more positive potentials. Surprisingly, it also completely abolished use-dependent slow inactivation. We showed previously that the equivalent residue in NaChBac (Asp60) interacts with the R3 gating charge in the S4 segment during activation using the disulfide locking method (DeCaen et al, PNAS 2008 105 (39) 15142-15147). To test whether this interaction between R3 and N49 was critical for slow inactivation, we mutated NavAb R3 to Cys. The resulting mutant NavAb_R3C also had positively shifted channel activation (+75 mV) and no use-dependent slow inactivation. The fact that these reciprocal mutations have the same functional effects suggests that interaction between R3 in the S4 segment and N49 in the S2 segment is an important link that stabilizes the activated state of the voltage sensor and triggers the slow inactivation process.

Crystal structure of a voltage-gated sodium channel in two potentially inactivated states.

In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2 Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.

Effects of lactate on the voltage-gated sodium channels of rat skeletal muscle: modulating current opinion

During muscle contraction, lactate production and translocation across the membrane increase. While it has recently been shown that lactate anion acts on chloride channel, less is known regarding a potential effect on the voltage-gated sodium channel (Na(v)) of skeletal muscle. The electrophysiological properties of muscle Na(v) were studied in the absence and presence of lactate (10 mM) by using the macropatch-clamp method in dissociated fibers from rat peroneus longus (PL). Lactate in the external medium (petri dish + pipette) increases the maximal sodium current, while the voltage dependence of activation and fast inactivation are shifted toward the hyperpolarized potentials. Lactate induces a leftward shift in the relationship between the kinetic parameters and the imposed potentials, resulting in an earlier recruitment of muscle Na(v). In addition, lactate significantly decreases the time constant of activation at voltages more negative than -10 mV, corresponding to an acceleration of Na(v) activation. The slow inactivation process is decreased by lactate, corresponding to an enhancement in the number of excitable Na(v). In an additional series of experiments, lactate (10 mM) was only added to the petri dish, while the pipette remained sealed on the membrane area. With this approach, the electrophysiological properties of Na(v) were unaffected by lactate compared with the control condition. Altogether, these data indicate that lactate modulates muscle Na(v) properties by an extracellular pathway. These effects are consistent with an enhancement in excitability, providing new insights into the role of lactate in muscle physiology.

Molecular Determinants of Slow Inactivation in Voltage-Gated Potassium Channels

Potassium channels respond to prolonged depolarizations with structural rearrangements that result in inactivated, nonconducting channels through a process termed slow inactivation. Significant efforts to understand this process have been made using structural, theoretical and experimental approaches yet the molecular details of slow inactivation in eukaryotic voltage-gated potassium (Kv) channels remain poorly understood. Data gleaned from prokaryotic (KcsA) and eukaryotic (Kv1.2/2.1) channels have implicated two adjacent and highly conserved aromatic side chains near the selectivity filter as critical determinants for slow inactivation. In particular, the indole nitrogens of both side chains, Trp434 and Trp435 in Shaker potassium channels, have been proposed to contribute to a hydrogen bond network that modulates slow inactivation, yet the direct demonstration of this notion is missing in Kv channels. Here we incorporate unnatural derivatives of Trp to directly demonstrate that the indole nitrogen of Trp434 is a crucial component of the slow inactivation process through two complementary substitutions. By subtly increasing the acidity of the indole nitrogen through fluorination (and therefore strengthening its hydrogen bond donor ability), the rate of slow inactivation was substantially decreased (4-fold slower than WT). Conversely, the novel unnatural amino acid side chain Ind, which lacks the indole nitrogen but is otherwise isosteric to Trp, increased the rate of inactivation more than 10-fold when Ind-containing channels were co-expressed with WT channels, as Ind-containing channels alone did not produce ionic current. In contrast to Trp434, the indole nitrogen of Trp435 does not contribute to slow inactivation as even relatively nonconservative mutations to Phe or Tyr at this site do not affect slow inactivation. Taken together, these results directly demonstrate the functional importance of the H-bonding ability of Trp434 in open pore stability in Kv channels.

Merging Structural Motifs of Functionalized Amino Acids and α-Aminoamides Results in Novel Anticonvulsant Compounds with Significant Effects on Slow and Fast Inactivation of Voltage-Gated Sodium Channels and in the Treatment of Neuropathic Pain

We recently reported that merging key structural pharmacophores of the anticonvulsant drugs lacosamide (a functionalized amino acid) with safinamide (an α-aminoamide) resulted in novel compounds with anticonvulsant activities superior to that of either drug alone. Here, we examined the effects of six such chimeric compounds on Na(+)-channel function in central nervous system catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that these compounds affected Na(+) channel fast and slow inactivation processes. Detailed electrophysiological characterization of two of these chimeric compounds that contained either an oxymethylene ((R)-7) or a chemical bond ((R)-11) between the two aromatic rings showed comparable effects on slow inactivation, use-dependence of block, development of slow inactivation, and recovery of Na(+) channels from inactivation. Both compounds were equally effective at inducing slow inactivation; (R)-7 shifted the fast inactivation curve in the hyperpolarizing direction greater than (R)-11, suggesting that in the presence of (R)-7, a larger fraction of the channels are in an inactivated state. None of the chimeric compounds affected veratridine- or KCl-induced glutamate release in neonatal cortical neurons. There was modest inhibition of KCl-induced calcium influx in cortical neurons. Finally, a single intraperitoneal administration of (R)-7, but not (R)-11, completely reversed mechanical hypersensitivity in a tibial-nerve injury model of neuropathic pain. The strong effects of (R)-7 on slow and fast inactivation of Na(+) channels may contribute to its efficacy and provide a promising novel therapy for neuropathic pain, in addition to its antiepileptic potential.

Development and Characterization of Novel Derivatives of the Antiepileptic Drug Lacosamide That Exhibit Far Greater Enhancement in Slow Inactivation of Voltage-Gated Sodium Channels

The novel antiepileptic drug, (R)-N-benzyl 2-acetamido-3-methoxypropionamide ((R)-lacosamide, Vimpat(®) ((R)-1)), was recently approved in the US and Europe for adjuvant treatment of partial-onset seizures in adults. (R)-1 preferentially enhances slow inactivation of voltage-gated Na(+) currents, a pharmacological process relevant in the hyperexcitable neuron. We have advanced a strategy to identify lacosamide binding partners by attaching affinity bait (AB) and chemical reporter (CR) groups to (R)-1 to aid receptor detection and isolation. We showed that select lacosamide AB and AB&CR derivatives exhibited excellent activities similar to (R)-1 in the maximal electroshock seizure model in rodents. Here, we examined the effect of these lacosamide AB and AB&CR derivatives and compared them with (R)-1 on Na(+) channel function in CNS catecholaminergic (CAD) cells. Using whole-cell patch clamp electrophysiology, we demonstrated that the test compounds do not affect the Na(+) channel fast inactivation process, that they were far better modulators of slow inactivation than (R)-1, and that modulation of the slow inactivation process was stereospecific. The lacosamide AB agents that contained either an electrophilic isothiocyanate ((R)-5) or a photolabile azide ((R)-8) unit upon AB activation gave modest levels of permanent Na(+) channel slow inactivation, providing initial evidence that these compounds may have covalently reacted with their cognate receptor(s). Our findings support the further use of these agents to delineate the (R)-1-mediated Na(+) channel slow inactivation process.

CaMKII regulation of voltage-gated sodium channels and cell excitability

i e P Sodium channels: Structure and function Voltage-gated sodium (Na) channels are central to cardiac excitation–contraction coupling (Figure 1) and thus myocardial contraction. They are involved in shaping the action potential waveform and in the conduction of electrical activity across the myocardium. At resting membrane potenials, cardiac Na channels are in the closed-available resting tate. During depolarization of the cell membrane, the chanels quickly activate, leading to influx of Na. Subsequent to hannel activation, the channels enter the inactivated state, hich is nonconductive for Na ions. For Na channel inacivation, a set of multiple processes occur, each of which is istinguishable by its recovery kinetic at strongly negative otentials. Fast inactivation occurs over tens of millisecnds, whereas intermediate or slow inactivation processes ccur with much slower kinetics in the range of hundreds of illiseconds or tens of seconds, respectively. An ultraslow omponent of Na current inactivation from the open state several hundreds of milliseconds) has been shown to conribute to action potential prolongation and early afterdepoarizations. This current seems to play an important role in eart failure. It was named late INa. Na channels (molecular weight 220 kDa) consist of various subunits with a required pore-forming -subunit for fundamental function. The Nav1.5 isoform (SCN5A) is the redominant isoform in the heart. However, other isoforms e.g., Nav1.1, Nav1.3, and Nav1.6) are also found in the heart. Auxiliary -subunits can modulate Na channel gating nd expression. They constitute only 10% of the molec-