Channel Activation Kinetics Research Articles

Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa.

Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K+ channels. We postulate that this effect might involve the inhibition of the large-conductance voltage- and Ca2+-operated K+ channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

Identifying the CaV1.1 voltage-sensing domains involved in channel gating and excitation contraction coupling.

Skeletal muscle CaV1.1 is a voltage gated calcium channel, which primarily functions as voltage sensor of excitation-contraction coupling (ECC). Upon depolarization, CaV1.1 triggers the opening of the RyR1 in the sarcoplasmic reticulum, causing the calcium release event essential for skeletal muscle contraction. Upon strong stimulation, CaV1.1 elicits a calcium current with kinetics and voltage-dependence different from those of ECC. Probably, these distinct activation properties arise from the differential roles of the four CaV1.1 voltage sensing domains (VSDs). Each VSD comprises of four transmembrane helices (S1-S4); S4 segments contain 4-5 positive charges, which interact with negative countercharges in the surrounding helices. These transient ion-pair interactions facilitate and tune the S4 motion in response to changes of the membrane potential. Using structure-guided mutagenesis with patch-clamp analysis and fluorescence calcium recordings, our group identified crucial countercharges in VSD I and IV able to regulate the channel's voltage sensitivity (I and IV) and activation kinetics (IV), without interfering with ECC. However, analogous mutations in VSDs II and III showed little or no effects on either channel gaiting or ECC, corroborating the notion that the four VSDs use different molecular mechanism for regulating their activation properties. Recently, two independent voltage-clamp fluorometry studies suggested that either VSD II or III is the main activator of ECC, based on their specific kinetics and voltage sensitivity matching those of ECC. While both studies agree that perhaps only a single VSD may activate ECC, they disagree on which one that might be. Here we address this problem using site-directed mutagenesis and chimeric CaV1.1 channels expressed in dysgenic myotubes. Constructs shifting the voltage-dependence of either ECC or channel activation reveal the specific contributions of the mutated VSDs in one or the other function of CaV1.1.

Function modulation of MDSC mHv1 channel by N terminal length.

Voltage-gated proton channel (Hv1) regulate the immunosuppressive function of myeloid-derived suppressor cells (MDSC) in mice. We asked if there were Hv1 isoforms present in MDSC. A prediction made by the NCBI showed that there could be longer isoforms of mouse Hv1, of 42 and four extra amino acid residues, called mHv1.2 and mHv1.3, respectively. These additional residues are predicted to express at the very beginning of the N terminal domain. We validated the prediction in vitro by RT-PCR, founding two isoforms of mHv1 and cloned them. We expressed these isoforms into Xenopus laevis oocytes for proton current recording studies by macropatch voltage clamp. All these isoforms studied satisfy the typical proton channel properties regarding the selectivity and channel activation kinetics. However, these isoforms exhibited clear differences both in their voltage-dependent gating and kinetics: the longer isoform, mHv1.2 exhibited the most right-displaced V0.5, followed by mHv1.3 and mHv1.1, while the most accelerated opening kinetics belonged to mHv1.1, followed by mHv1.3 and finally mHv1.2. We studied the unitary conductance of these isoforms by non-stationary noise analysis, evidencing values related to the fS order. All the cloned isoforms are expressed in the membrane as dimers, as shown by the estimation of the charge coupled to the opening by limit slope technique. These results strongly suggest that all these isoforms were typical Hv1 channels that possess unique biophysical properties. Since the different isoforms exhibited exclusively differences in their N terminal, the longer the N terminal length, the slower the opening kinetics and the shifted to the right the GV curve. We propose that the differences in the voltage-dependent gating exhibited by the isoforms are due by the extension of their N terminal.

Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage (V 1/2) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized V 1/2 in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents.SIGNIFICANCE STATEMENT Vestibular afferents express a diverse complement of ion channels. In vitro studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Long QT syndrome-associated calmodulin mutations and their interactions with the Kv7.1 voltage-gated potassium channel

Abstract Introduction Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+) dependent signalling. Its flexible structure allows CaM to bind and modulate many targets, including cardiac ion channels. Genotyping has revealed several CaM mutations associated with congenital disorders of heart rhythm, known as long QT-syndrome (LQTS). LQTS patients suffer from prolonged ventricular recovery times (QT-interval) which increases their risk of significant cardiac events. Loss of function KV7.1 mutations are the largest cause of LQTS, accounting for >50% of cases. CaM facilitates Ca2+-sensitivity to KV7.1 in producing IKs, Kv7.1 mutations which reduce CaM binding promote LQTS pathology. However, the effects of LQTS-associated CaM mutations on Kv7.1 function remain unknown. Purpose To determine the biophysical consequences of congenital LQTS-associated CaM mutations and how they alter modulation of Kv7.1 in producing the ventricular repolarising IKs current. Methods WT and mutant CaM proteins were recombinantly expressed and purified for biophysical characterisation. Using circular dichroism, secondary structures and thermostability of proteins were quantified. Isothermal titration calorimetry was used to quantitatively measure interactions between CaM proteins and binding sites of KV7.1 (Helix B). NMR was employed to study the conformations of target-bound WT and mutant proteins. Whole cell currents were determined using voltage clamp electrophysiology in HEK cells. Results Mutations significantly changed the thermostability and secondary structure distributions of CaM, and also caused site-dependent increases in susceptibility to protease digestion. CaM interacted with Helix B (KV7.1) via both Ca2+-dependent and independent mechanisms. Ca2+ dependent binding to Helix B was much higher affinity than Ca2+-independent binding, with >2000-fold reduction in dissociation constant measured. LQTS-CaM variants reduced Helix B affinity with the largest reductions found in EF-hand IV mutants. These mutants also adopted most distinct conformations when Helix B-bound. Calmodulation of the KV7.1 channel produced larger (IKs) currents without altering channel activation kinetics. IKs exhibited Ca2+-sensitivity, in response to increased cytosolic Ca2+, larger currents were generated. Modulation by CaM mutants reduced current density at systolic Ca2+-concentrations (1000 nM), within physiological time periods (0.35 s), revealing a direct QT-prolonging modulatory effect. Conclusions Provided here are mechanistic insights as to how LQTS-associated CaM variants contribute to electrical disease of the heart. Mutations in the highly conserved structure of CaM disrupt protein conformation and perturb complex formation with KV7.1. This results in aberrant Ca2+-sensitivity of Kv7.1, reducing IKs generation. This ultimately decreases the repolarisation capacity of cells and would extend the QT interval of myocytes. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation Intermediate Basic Science Research Fellowship

Investigation of novel de novo KCNC2 variants causing severe developmental and early-onset epileptic encephalopathy

Purpose The voltage-gated potassium channel Kv3.2, encoded by KCNC2, facilitates fast-spiking GABAergic interneurons to fire action potentials at high frequencies. It is pivotal to maintaining excitation/inhibition balance in mammalian brains. This study identified two novel de novo KCNC2 variants, p.Pro470Ser (P470S) and p.Phe382Leu (F382L), in patients with early onset developmental and epileptic encephalopathy (DEE).Methods To examine the molecular basis of DEE, we studied the functional characteristics of variant channels using patch-clamp techniques and computational modeling.Results Whole-cell patch clamp recordings from infected HEK293 cells revealed that channel activation and deactivation kinetics strongly decreased in both Kv3.2 P470S and F382L variant channels. This decrease also occurred in Kv3.2 p.Val471Leu (V471L) channels, known to be associated with DEE. In addition, Kv3.2 F382L and V471L variants exhibited a significant increase in channel conductance and a ∼20 mV negative shift in the threshold for voltage-dependent activation. Simulations of model GABAergic interneurons revealed that all variants decreased neuronal firing frequency. Thus, the variants’ net loss-of-function effects disinhibited neural networks.Conclusion Our findings provide compelling evidence supporting the role of KCNC2 as a disease-causing gene in human neurodevelopmental delay and epilepsy.

NaV1.9 current in muscle afferent neurons is enhanced by substances released during muscle activity.

Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NaV) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NaV currents. The rise time of this enhancement averaged ∼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NaV1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NaV current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NaV1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NaV1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NaV1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NaV1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.

Effect of an autism-associated KCNMB2 variant, G124R, on BK channel properties.

BK K+ channels are critical regulators of neuron and muscle excitability, comprised of a tetramer of pore-forming αsubunits from the KCNMA1 gene and cell- and tissue-selective β subunits (KCNMB1-4). Mutations in KCNMA1 are associated with neurological disorders, including autism. However, little is known about the role of neuronal BK channel β subunits in human neuropathology. The β2 subunit is expressed in central neurons and imparts inactivation to BK channels, as well as altering activation and deactivation gating. In this study, we report the functional effect of G124R, a novel KCNMB2 mutation obtained from whole-exome sequencing of a patient diagnosed with autism spectrum disorder. Residue G124, located in the extracellular loop between TM1 and TM2, is conserved across species, and the G124R missense mutation is predicted deleterious with computational tools. To investigate the pathogenicity potential, BK channels were co-expressed with β2WT and β2G124R subunits in HEK293T cells. BK/β2 currents were assessed from inside-out patches under physiological K+ conditions (140/6 mM K+ and 10 μM Ca2+) during activation and inactivation (voltage-dependence and kinetics). Using β2 subunits lacking inactivation (β2IR) revealed that currents from BK/β2IRG124R channels activated 2-fold faster and deactivated 2-fold slower compared with currents from BK/β2IRWT channels, with no change in the voltage-dependence of activation (V1/2). Despite the changes in the BK channel opening and closing, BK/β2G124R inactivation rates (τinact and τrecovery), and the V1/2 of inactivation, were unaltered compared with BK/β2WT channels under standard steady-state voltage protocols. Action potential-evoked current was also unchanged. Thus, the mutant phenotype suggests the β2G124R TM1-TM2 extracellular loop could regulate BK channel activation and deactivation kinetics. However, additional evidence is needed to validate pathogenicity for this patient-associated variant in KCNMB2.

Stimulant Drugs of Abuse and Cardiac Arrhythmias.

Nonmedical use of prescription and nonprescription drugs is a worldwide epidemic, rapidly growing in magnitude with deaths because of overdose and chronic use. A vast majority of these drugs are stimulants that have various effects on the cardiovascular system including the cardiac rhythm. Drugs, like cocaine and methamphetamine, have measured effects on the conduction system and through several direct and indirect pathways, utilizing multiple second messenger systems, change the structural and electrical substrate of the heart, thereby promoting cardiac dysrhythmias. Substituted amphetamines and cocaine affect the expression and activation kinetics of multiple ion channels and calcium signaling proteins resulting in EKG changes, and atrial and ventricular brady and tachyarrhythmias. Preexisting conditions cause substrate changes in the heart, which decrease the threshold for such drug-induced cardiac arrhythmias. The treatment of cardiac arrhythmias in patients who take drugs of abuse may be specialized and will require an understanding of the unique underlying mechanisms and necessitates a multidisciplinary approach. The use of primary or secondary prevention defibrillators in drug abusers with chronic systolic heart failure is both sensitive and controversial. This review provides a broad overview of cardiac arrhythmias associated with stimulant substance abuse and their management.

Hypoxic Regulation of the Large-Conductance, Calcium and Voltage-Activated Potassium Channel, BK

Hypoxia is a condition characterized by a reduction of cellular oxygen levels derived from alterations in oxygen balance. Hypoxic events trigger changes in cell-signaling cascades, oxidative stress, activation of pro-inflammatory molecules, and growth factors, influencing the activity of various ion channel families and leading to diverse cardiovascular diseases such as myocardial infarction, ischemic stroke, and hypertension. The large-conductance, calcium and voltage-activated potassium channel (BK) has a central role in the mechanism of oxygen (O2) sensing and its activity has been related to the hypoxic response. BK channels are ubiquitously expressed, and they are composed by the pore-forming α subunit and the regulatory subunits β (β1–β4), γ (γ1–γ4), and LINGO1. The modification of biophysical properties of BK channels by β subunits underly a myriad of physiological function of these proteins. Hypoxia induces tissue-specific modifications of BK channel α and β subunits expression. Moreover, hypoxia modifies channel activation kinetics and voltage and/or calcium dependence. The reported effects on the BK channel properties are associated with events such as the increase of reactive oxygen species (ROS) production, increases of intracellular Calcium ([Ca2+]i), the regulation by Hypoxia-inducible factor 1α (HIF-1α), and the interaction with hemeproteins. Bronchial asthma, chronic obstructive pulmonary diseases (COPD), and obstructive sleep apnea (OSA), among others, can provoke hypoxia. Untreated OSA patients showed a decrease in BK-β1 subunit mRNA levels and high arterial tension. Treatment with continuous positive airway pressure (CPAP) upregulated β1 subunit mRNA level, decreased arterial pressures, and improved endothelial function coupled with a reduction in morbidity and mortality associated with OSA. These reports suggest that the BK channel has a role in the response involved in hypoxia-associated hypertension derived from OSA. Thus, this review aims to describe the mechanisms involved in the BK channel activation after a hypoxic stimulus and their relationship with disorders like OSA. A deep understanding of the molecular mechanism involved in hypoxic response may help in the therapeutic approaches to treat the pathological processes associated with diseases involving cellular hypoxia.

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels.

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (NaV) and K (KV) channels. NaV channels activate much faster and allow the initial Na+ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in KV channels but an equally highly conserved threonine in NaV channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker KV channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of NaV channels comes from a stronger dielectric polarization by threonine (NaV channel) produced as the first gating charge enters the gating pore compared with isoleucine (KV channel).

Estimation of the Motor Threshold for Near-Rectangular Stimuli Using the Hodgkin-Huxley Model.

The motor threshold measurement is a standard in preintervention probing in TMS experiments. We aim to predict the motor threshold for near-rectangular stimuli to efficiently determine the motor threshold size before any experiments take place. Estimating the behavior of large-scale networks requires dynamically accurate and efficient modeling. We utilized a Hodgkin–Huxley (HH) type model to evaluate motor threshold values and computationally validated its function with known true threshold data from 50 participants trials from state-of-the-art published datasets. For monophasic, bidirectional, and unidirectional rectangular stimuli in posterior-anterior or anterior-posterior directions as generated by the cTMS device, computational modeling of the HH model captured the experimentally measured population-averaged motor threshold values at high precision (maximum error ≤ 8%). The convergence of our biophysically based modeling study with experimental data in humans reveals that the effect of the stimulus shape is strongly correlated with the activation kinetics of the voltage-gated ion channels. The proposed method can reliably predict motor threshold size using the conductance-based neuronal models and could therefore be embedded in new generation neurostimulators. Advancements in neural modeling will make it possible to enhance treatment procedures by reducing the number of delivered magnetic stimuli to participants.

An Introduction to Patch Clamp Recording.

Electrophysiology is an essential tool aiding the study of the functions and dysfunctions of electrically excitable cells and their networks. The patch clamp method is a refined electrophysiological technique that can directly measure the membrane potential and/or theamount of current passing acrossthe cell membrane. The patch clamp techniqueis also incredibly versatile and can be used in a variety of different configurations to study a range of properties,from spontaneous cell firing activity in native tissue to the activation and/or deactivation kinetics of individual channels expressed in recombinant cell lines. In this chapter we give an overview of patch clamping and how the different configurations can be set up and applied to electrophysiologicalresearch.

Disease-associated HCN4 V759I variant is not sufficient to impair cardiac pacemaking

The hyperpolarization-activated cation current If is a key determinant for cardiac pacemaker activity. It is conducted by subunits of the hyperpolarization-activated cyclic nucleotide–gated (HCN) channel family, of which HCN4 is predominant in mammalian heart. Both loss-of-function and gain-of-function mutations of the HCN4 gene are associated with sinus node dysfunction in humans; however, their functional impact is not fully understood yet. Here, we sought to characterize a HCN4 V759I variant detected in a patient with a family history of sick sinus syndrome. The genomic analysis yielded a mono-allelic HCN4 V759I variant in a 49-year-old woman presenting with a family history of sick sinus syndrome. This HCN4 variant was previously classified as putatively pathogenic because genetically linked to sudden infant death syndrome and malignant epilepsy. However, detailed electrophysiological and cell biological characterization of HCN4 V759I in Xenopus laevis oocytes and embryonic rat cardiomyocytes, respectively, did not reveal any obvious abnormality. Voltage dependence and kinetics of mutant channel activation, modulation of cAMP-gating by the neuronal HCN channel auxiliary subunit PEX5R, and cell surface expression were indistinguishable from wild-type HCN4. In good agreement, the clinically likewise affected mother of the patient does not exhibit the reported HCN4 variance. HCN4 V759I resembles an innocuous genetic HCN channel variant, which is not sufficient to disturb cardiac pacemaking. Once more, our work emphasizes the importance of careful functional interpretation of genetic findings not only in the context of hereditary cardiac arrhythmias.

Low-Resistance silver bromide electrodes for recording fast ion channel kinetics under voltage clamp conditions

BackgroundTwo-electrode voltage clamp is a widely used technique for studying ionic currents. However, fast activation kinetics of ion channels are disguised by the capacitive transient during voltage clamp of Xenopus oocytes. The limiting factors of clamp performance include, but are not limited to, amplifier gain, membrane capacitance, and micropipette resistance. Previous work has focused on increasing amplifier gain (e.g.; high performing two-electrode amplifiers) or reducing the membrane capacitance (e.g.; the cut-open technique). New methodThe use of an Ag-AgBr electrode with saturated KBr solution to reduce micropipette resistance. ResultsThe conductivity of 4 M KBr was 37 % higher compared to 3 M KCl and the micropipette resistance was reduced by 19 % when 4 M KBr was used, compared to the standard 3 M KCl solution. Micropipette resistances correlated positively with capacitive transient durations. Neither the current-voltage relationship of the voltage-gated sodium channel, Nav1.7, nor Xenopus oocyte stability were affected by bromide ions. Comparison with existing methodsThe de facto standard for two-electrode voltage clamp is 3 M KCl and Ag-AgCl electrodes, which are associated an unnecessarily high micropipette resistance. Elsewise, cut-open voltage clamp techniques are technically demanding and require manipulation of the intracellular environment. ConclusionsThe use of an Ag-AgBr electrode with saturated KBr as micropipette solution reduces the capacitive transient in two-electrode voltage clamp recordings. Moreover, the exchange of chloride against bromide ions does not seem to affect oocyte physiology and ion channel kinetics.

Two Novel Peptide Toxins from the Spider Cyriopagopus longipes Inhibit Tetrodotoxin-Sensitive Sodium Channels.

Sodium channels play a critical role in the generation and propagation of action potentials in excitable tissues, such as nerves, cardiac muscle, and skeletal muscle, and are the primary targets of toxins found in animal venoms. Here, two novel peptide toxins (Cl6a and Cl6b) were isolated from the venom of the spider Cyriopagopus longipes and characterized. Cl6a and Cl6b were shown to be inhibitors of tetrodotoxin-sensitive (TTX-S), but not TTX-resistant, sodium channels. Among the TTX-S channels investigated, Cl6a and Cl6b showed the highest degree of inhibition against NaV1.7 (half-maximal inhibitory concentration (IC50) of 11.0 ± 2.5 nM and 18.8 ± 2.4 nM, respectively) in an irreversible manner that does not alter channel activation, inactivation, or repriming kinetics. Moreover, analysis of NaV1.7/NaV1.8 chimeric channels revealed that Cl6b is a site 4 neurotoxin. Site-directed mutagenesis analysis indicated that D816, V817, and E818 observably affected the efficacy of the Cl6b-NaV1.7 interaction, suggesting that these residues might directly affect the interaction of NaV1.7 with Cl6b. Taken together, these two novel peptide toxins act as potent and sustained NaV1.7 blockers and may have potential in the pharmacological study of sodium channels.

Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions

Invertebrate LCaV3 shares the quintessential features of vertebrate CaV3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca2+ channels, the CaV1 and CaV2 channels. Unlike the vertebrates though, CaV3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCaV3 channel into a higher Na+ and lower Ca2+ current passing channel, more resembling a classical NaV1 Na+ channel. Cnidarian CaV3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates. We illustrate here that the preferences for greater Na+ or Ca2+ ion current passing through CaV3 T-type channels are contributed by paired cysteines within D2L5 and D4L6 extracellular loops looming above the pore selectivity filter. Swapping of invertebrate tri- and tetra-cysteine containing extracellular loops, generates higher Na+ current passing channels in human CaV3.2 channels, while corresponding mono- and di-cysteine loop pairs in human CaV3.2 generates greater Ca2+ current passing, invertebrate LCaV3 channels. Alanine substitutions of unique D2L5 loop cysteines of LCaV3 channels increases relative monovalent ion current sizes and increases the potency of Zn2+ and Ni2+ block by ~ 50× and ~ 10× in loop cysteine mutated channels respectively, acquiring characteristics of the high affinity block of CaV3.2 channels, including the loss of the slowing of inactivation kinetics during Zn2+ block. Charge neutralization of a ubiquitous aspartate residue of calcium passing CaV1, CaV2 and CaV3 channels, in the outer pore of the selectivity filter residues in Domain II generates higher Na+ current passing channels in a manner that may resemble how the unique D2L5 extracellular loops of invertebrate CaV3 channels may confer a relatively higher peak current size for Na+ ions over Ca2+ The extracellular loops of CaV3 channels are not engaged with accessory subunit binding, as the other Na+ (NaV1) and Ca2+ (CaV1/CaV2) channels, enabling diversity and expansion of cysteine-bonded extracellular loops, which appears to serve, amongst other possibilities, to alter to the preferences for passage of Ca2+ or Na+ ions through invertebrate CaV3 channels.

Ca2+ entry through NaV channels generates submillisecond axonal Ca2+ signaling.

Calcium ions (Ca2+) are essential for many cellular signaling mechanisms and enter the cytosol mostly through voltage-gated calcium channels. Here, using high-speed Ca2+ imaging up to 20 kHz in the rat layer five pyramidal neuron axon we found that activity-dependent intracellular calcium concentration ([Ca2+]i) in the axonal initial segment was only partially dependent on voltage-gated calcium channels. Instead, [Ca2+]i changes were sensitive to the specific voltage-gated sodium (NaV) channel blocker tetrodotoxin. Consistent with the conjecture that Ca2+ enters through the NaV channel pore, the optically resolved ICa in the axon initial segment overlapped with the activation kinetics of NaV channels and heterologous expression of NaV1.2 in HEK-293 cells revealed a tetrodotoxin-sensitive [Ca2+]i rise. Finally, computational simulations predicted that axonal [Ca2+]i transients reflect a 0.4% Ca2+ conductivity of NaV channels. The findings indicate that Ca2+ permeation through NaV channels provides a submillisecond rapid entry route in NaV-enriched domains of mammalian axons.

Analysis of tetrodotoxin-sensitive sodium and low voltage-activated calcium channels in developing mouse retinal horizontal cells

Expression patterns of voltage-gated ion channels determine the spatio-temporal dynamics of ion currents that supply excitable neurons in developing tissue with proper electrophysiological properties. The purpose of the study was to identify fast cationic inward currents in mouse retinal horizontal cells (HCs) and describe their biophysical properties at different developmental stages. We also aimed to reveal their physiological role in shaping light responses (LRs) in adult HCs. HCs were recorded in horizontal slices of wild-type mouse retina at postnatal stages ranging from p8 through p60. Voltage-dependent inward currents were isolated with appropriate voltage protocols and blockers specific for sodium and T-type calcium channels. LRs were evoked with full-field flashes (130 μW/cm2). Transient and steady inward currents were identified at all developmental stages. Transient currents were mediated by T-type calcium and TTX-sensitive sodium channels, whereas steady currents were blocked by cadmium, indicating the presence of high voltage-activated calcium channels. Activation and steady-state inactivation kinetics of T-type calcium channels revealed a contribution to the resting membrane potential during postnatal development. Additionally, both sodium and T-type calcium channels had an impact on HC LRs at light offset in adult animals. Our results showed that the voltage-dependent inward currents of postnatally developing mouse HCs consist of T-type calcium, TTX-sensitive sodium, and high voltage-activated calcium channels, and that transient ionic currents contributed to light-evoked responses of adult HCs, suggesting a role in HC information processing.

Temperature Dependence of the Biophysical Mechanisms Underlying the Inhibition and Enhancement Effect of Amiodarone on hERG Channels.

hERG K+ channel is important for controlling the duration of cardiac action potentials. Amiodarone (AMD), a widely prescribed class III antiarrhythmic, could inhibit hERG currents with relatively few tachyarrhythmic adverse events. We use injected Xenopus oocyte with two-electrode voltage clamp techniques to characterize the action of AMD on hERG channels. We found that AMD binds to the resting hERG channel with an apparent dissociation constant of ∼1.4 μM, and inhibits hERG currents at mild and strong depolarization pulses by slowing activation and enhancing inactivation, respectively, at 22°C. The activation kinetics of hERG channel activation are much faster, but inactivation kinetics are slower at 37°C. AMD accordingly has a 15% to 20% weaker and stronger inhibitory effect at mild and strong depolarization (e.g., -60 and +30 mV, 0.3-second pulse), respectively. In the meanwhile, the resurgent hERG tail currents are dose-dependently inhibited by AMD without altering the kinetics of current decay at both 22°C and 37°C, indicating facilitation of recovery from inactivation via the silent route. Most importantly, AMD no longer inhibits but enhances hERG currents at a mild pulse shortly after a prepulse at 37°C, but not so much at 22°C. We conclude that AMD is an effective hERG channel-gating modifier capable of lengthening the plateau phase of cardiac action potential (without increasing the chance of afterdepolarization). AMD, however, should be used with caution in hypothermia or the other scenarios that slow hERG channel activation. SIGNIFICANCE STATEMENT: It is known that amiodarone (AMD) acts on hERG K+ channels to treat cardiac arrhythmias with relatively little arrhythmogenicity. We found that AMD enhances hERG channel inactivation but slows activation as well as recovery from inactivation, and thus has a differential inhibition and enhancement effect on hERG currents at different phases of membrane voltage changes, especially at 37°C, but not so much at 22°C. AMD is therefore a relatively ideal agent against tachyarrhythmia at 37°C, but should be more cautiously used at lower temperatures or relevant pathophysiological/pharmacological scenarios associated with slower hERG channel activation because of the increased chances of adverse events.