Gating Properties Of Channels Research Articles

The biophysical properties of TRIC-A and TRIC-B and their interactions with RyR2.

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are thought to provide counter-ion currents to enable charge equilibration across the sarco/endoplasmic reticulum (SR) and nuclear membranes. However, there is also evidence that TRIC-A may interact directly with ryanodine receptor type 1 (RyR1) and 2 (RyR2) to alter RyR channel gating. It is therefore possible that the reverse is also true, where the presence of RyR channels is necessary for fully functional TRIC channels. We therefore coexpressed mouse TRIC-A or TRIC-B with mouse RyR2 in HEK293 cells to examine if after incorporating membrane vesicles from these cells into bilayers, the presence of TRIC affects RyR2 function, and to characterize the permeability and gating properties of the TRIC channels. Importantly, we used no purification techniques or detergents to minimize damage to TRIC and RyR2 proteins. We found that both TRIC-A and TRIC-B altered the gating behavior of RyR2 and its response to cytosolic Ca2+ but that TRIC-A exhibited a greater ability to stimulate the opening of RyR2. Fusing membrane vesicles containing TRIC-A or TRIC-B into bilayers caused the appearance of rapidly gating current fluctuations of multiple amplitudes. The reversal potentials of bilayers fused with high numbers of vesicles containing TRIC-A or TRIC-B revealed both Cl- and K+ fluxes, suggesting that TRIC channels are relatively non-selective ion channels. Our results indicate that the physiological roles of TRIC-A and TRIC-B may include direct, complementary regulation of RyR2 gating in addition to the provision of counter-ion currents of both cations and anions.

Incorporating physics to overcome data scarcity in predictive modeling of protein function: A case study of BK channels.

Machine learning has played transformative roles in numerous chemical and biophysical problems such as protein folding where large amount of data exists. Nonetheless, many important problems remain challenging for data-driven machine learning approaches due to the limitation of data scarcity. One approach to overcome data scarcity is to incorporate physical principles such as through molecular modeling and simulation. Here, we focus on the big potassium (BK) channels that play important roles in cardiovascular and neural systems. Many mutants of BK channel are associated with various neurological and cardiovascular diseases, but the molecular effects are unknown. The voltage gating properties of BK channels have been characterized for 473 site-specific mutations experimentally over the last three decades; yet, these functional data by themselves remain far too sparse to derive a predictive model of BK channel voltage gating. Using physics-based modeling, we quantify the energetic effects of all single mutations on both open and closed states of the channel. Together with dynamic properties derived from atomistic simulations, these physical descriptors allow the training of random forest models that could reproduce unseen experimentally measured shifts in gating voltage, ∆V1/2, with a RMSE ~ 32 mV and correlation coefficient of R ~ 0.7. Importantly, the model appears capable of uncovering nontrivial physical principles underlying the gating of the channel, including a central role of hydrophobic gating. The model was further evaluated using four novel mutations of L235 and V236 on the S5 helix, mutations of which are predicted to have opposing effects on V1/2 and suggest a key role of S5 in mediating voltage sensor-pore coupling. The measured ∆V1/2 agree quantitatively with prediction for all four mutations, with a high correlation of R = 0.92 and RMSE = 18 mV. Therefore, the model can capture nontrivial voltage gating properties in regions where few mutations are known. The success of predictive modeling of BK voltage gating demonstrates the potential of combining physics and statistical learning for overcoming data scarcity in nontrivial protein function prediction.

Differential regulation of tetramerization of the AMPA receptor glutamate–gated ion channel by auxiliary subunits

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

Inhibition of voltage-dependent K+ currents of rabbit coronary arterial smooth muscle cells by the atypical antipsychotic paliperidone.

Paliperidone, an atypical antipsychotic, is widely used to treat schizophrenia. In this study, we explored whether paliperidone inhibited the voltage-dependent K+ (Kv) channels of rabbit coronary arterial smooth muscle cells. Paliperidone reduced Kv channel activity in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50 ) of 16.58 ± 3.03 μM and a Hill coefficient of 0.60 ±0.04. It did not significantly shift the steady-state activation or inactivation curves, suggesting that the drug did not affect the gating properties of Kv channels. In the presence of paliperidone, the application of 20 repetitive depolarizing pulses at 1 and 2Hz gradually increased the inhibition of the Kv current. Further, the recovery time constant after Kv channel inactivation was increased by paliperidone, indicating that it inhibited the Kv channel in a use (state)-dependent manner. Its inhibitory effects were reduced by pretreatment with a Kv1.5 subtype inhibitor. However, pretreatment with a Kv2.1 or Kv7 inhibitor did not reduce its inhibitory effect. We conclude that paliperidone inhibits Kv channels (mainly Kv1.5 subtype channels) in a concentration- and use (state)-dependent manner without changing channel gating.

PI(4,5)P2 regulates the gating of NaV1.4 channels.

Voltage-gated sodium (NaV) channels are densely expressed in most excitable cells and activate in response to depolarization, causing a rapid influx of Na+ ions that initiates the action potential. The voltage-dependent activation of NaV channels is followed almost instantaneously by fast inactivation, setting the refractory period of excitable tissues. The gating cycle of NaV channels is subject to tight regulation, with perturbations leading to a range of pathophysiological states. The gating properties of most ion channels are regulated by the membrane phospholipid, phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2). However, it is not known whether PI(4,5)P2 modulates the activity of NaV channels. Here, we utilize optogenetics to activate specific, membrane-associated phosphoinositide (PI)-phosphatases that dephosphorylate PI(4,5)P2 while simultaneously recording NaV1.4 channel currents. We show that dephosphorylating PI(4,5)P2 left-shifts the voltage-dependent gating of NaV1.4 to more hyperpolarized membrane potentials, augments the late current that persists after fast inactivation, and speeds the rate at which channels recover from fast inactivation. These effects are opposed by exogenous diC8PI(4,5)P2. We provide evidence that PI(4,5)P2 is a negative regulator that tunes the gating behavior of NaV1.4 channels.

Transient and Sustained Ganglion Cell Light Responses Are Differentially Modulated by Intrinsically Produced Reactive Oxygen Species Acting upon Specific Voltage-Gated Na+ Channel Isoforms.

Increasing spike rates drive greater neuronal energy demand. In turn, mitochondrial ATP production leads to the generation of reactive oxygen species (ROS) that can modulate ion channel gating. Does ROS production autoregulate the excitability of a neuron? We investigated the links between retinal ganglion cell (RGC) excitability and spike activity-driven ROS production in male and female mice. Changes to the light-evoked and current-evoked spike patterns of functionally identified αRGC subtypes, along with their NaV channel-gating properties, were recorded during experimentally induced decreases and increases of intracellular ROS. During periods of highest spike rates (e.g., following light onset in ON sustained RGCs and light offset in OFF sustained RGCs), these αRGC subtypes responded to reductions of ROS (induced by catalase or glutathione monoethyl ester) with higher spike rates. Increases in ROS (induced by mercaptosuccinate, antimycin-A, or H2O2) lowered spike rates. In ON and OFF transient RGCs, there were no changes in spike rate during ROS decreases but increased ROS increased spiking. This suggests that endogenous ROS are intrinsic neuromodulators in RGCs having high metabolic demands but not in RGCs with lower energy needs. We identified ROS-induced shifts in the voltage-dependent gating of specific isoforms of NaV channels that account for the modulation of ON and OFF sustained RGC spike frequency by ROS-mediated feedback. ROS-induced changes to NaV channel gating, affecting activation and inactivation kinetics, are consistent with the differing spike pattern alterations observed in RGC subtypes. Cell-autonomous generation of ROS during spiking contributes to tuning the spike patterns of RGCs.SIGNIFICANCE STATEMENT Energy production within retinal ganglion cells (RGCs) is accompanied by metabolic by-products harmful to cellular function. How these by-products modulate the excitability of RGCs bears heavily on visual function and the etiology of optic neuropathies. A novel hypothesis of how RGC metabolism can produce automodulation of electrical signaling was tested by identifying the characteristics and biophysical origins of changes to the excitability of RGCs caused by oxidizing by-products in the retina. This impacts our understanding of the pathophysiology of RGC dysfunction, supporting an emerging model in which increases in oxidizing chemical species during energy production, but not necessarily bioenergetic failure, lead to preferential degeneration of specific subtypes of RGCs, yielding loss of different aspects of visual capacity.

Voltage-dependent transitions between resting and activated states of the four Cav1.1 voltage sensors observed in MD simulation.

The sliding helix hypothesis describes how, upon depolarization and repolarization of the membrane, the positive gating charges of the S4 helices of voltage-sensing domains (VSDs) one by one move across the membrane electrical field and thereby effect the opening and closing of ion channels. Voltage-gated calcium channels contain four VSDs, which together regulate gating of a common pore. Although these four VSDs are highly homologous, they differ from each other, e.g. in the number of gating charges and in the number and position of their countercharges. Recent atomic-resolution structures of voltage-gated calcium and sodium channels delineate the endpoints of the VSD movement upon depolarization, showing the S4 helix in the activated up-state. While the first structures of VSDs in resting states have been solved for homotetrameric (prokaryotic) channels, resting state structures of the (pseudo-)heterotetrameric eukaryotic sodium and calcium channels are still lacking. Accordingly, structural models showing the transitions from the resting to the activated state and back are still elusive. Here we addressed this problem using enhanced sampling molecular dynamics simulations of the CaV1.1 calcium channel exposed to a strong membrane potential. Indeed, starting from the known up-state, the S4 helices of the four VSDs moved down to positions closely matching the experimentally determined resting state of the bacterial NaVAb channel. Importantly, the trajectories, the transiently formed ionic interactions between gating- and countercharges, and the speed of the S4 transitions differed substantially between the four VSDs. This is consistent with data from recent mutagenesis and voltage-clamp fluorometry studies on CaV1.1 and allows relating the individual VSD movements with their putative roles in the regulation of distinct channel gating properties, as well as their role in the activation of excitation-contraction coupling in skeletal muscle.

GJB2 and GJB6 gene transcripts in the human cochlea: A study using RNAscope, confocal, and super-resolution structured illumination microscopy.

BackgroundGap junction (GJ) proteins, connexin26 and 30, are highly prevalent in the human cochlea (HC), where they are involved in transcellular signaling, metabolic supply, and fluid homeostasis. Their genes, GJB2 and GJB6, are both located at the DFNB1 locus on chromosome 13q12. Mutations in GJB2 may cause mild to profound non-syndromic deafness. Here, we analyzed for the first time the various expressions of GJB2 and GJB6 gene transcripts in the different cell networks in the HC using the RNAscope technique.Materials and methodsArchival paraformaldehyde-fixed sections of surgically obtained HC were used to label single mRNA oligonucleotides using the sensitive multiplex RNAscope® technique with fluorescent-tagged probes. Positive and negative controls also included the localization of ATP1A1, ATP1A2, and KCNJ10 gene transcripts in order to validate the specificity of labeling.ResultsConfocal and super-resolution structured illumination microscopy (SR-SIM) detected single gene transcripts as brightly stained puncta. The GJB2 and GJB6 gene transcripts were distributed in the epithelial and connective tissue systems in all three cochlear turns. The largest number of GJB2 and GJB6 gene transcripts was in the outer sulcus, spiral ligament, and stria vascularis (SV). Oligonucleotides were present in the supporting cells of the organ of Corti (OC), spiral limbus fibrocytes, and the floor of the scala vestibuli. Multiplex gene data suggest that cells in the cochlear lateral wall contain either GJB2 or GJB6 gene transcripts or both. The GJB6, but not GJB2, gene transcripts were found in the intermediate cells but none were found in the marginal cells. There were no GJB2 or GJB6 gene transcripts found in the hair cells and only a few in the spiral ganglion cells.ConclusionBoth GJB2 and GJB6 mRNA gene transcripts were localized in cells in the adult HC using RNAscope® in situ hybridization (ISH) and high resolution microscopy. Generally, GJB6 dominated over GJB2, except in the basal cells. Results suggest that cells may contain either GJB2 or GJB6 gene transcripts or both. This may be consistent with specialized GJ plaques having separate channel permeability and gating properties. A reduction in the number of GJB2 gene transcripts was found in the basal turn. Such information may be useful for future gene therapy.

Characterization of two kdr mutations at predicted pyrethroid receptor site 2 in the sodium channels of Aedes aegypti and Nilaparvata lugens

Pyrethroid insecticides prolong the opening of insect sodium channels by binding to two predicted pyrethroid receptor sites (PyR), PyR1 and PyR2. Many naturally-occurring sodium channel mutations that confer pyrethroid resistance (known as knockdown resistance, kdr) are located at PyR1. Recent studies identified two new mutations, V253F and T267A, at PyR2, which co-exist with two well-known mutations F1534C or M918T, at PyR1, in pyrethroid-resistant populations of Aedes aegypti and Nilaparvata lugens, respectively. However, the role of the V253F and T267A mutations in pyrethroid resistance has not been functionally examined. Here we report functional characterization of the V253F and T267A mutations in the Ae. aegypti sodium channel AaNav2-1 and the N. lugens sodium channel NlNav1 expressed in Xenopus oocytes. Both mutations alone reduced channel sensitivity to pyrethroids, including etofenprox. We docked etofenprox in a homology model of the pore module of the NlNav1 channel based on the crystal structure of an open prokaryotic sodium channel NavMs. In the low-energy binding pose etofenprox formed contacts with V253, T267 and a previously identified L1014 within PyR2. Combining of V253F or T267A with F1534C or M918T results in a higher level of pyrethroid insensitivity. Furthermore, both V253F and T267A mutations altered channel gating properties. However, V253F- and T267A-induced gating modifications was not observed in the double mutant channels. Our findings highlight the first example in which naturally-found combinational mutations in PyR1 and PyR2 not only confer higher level pyrethroid insensitivity, but also reduce potential fitness tradeoff in pyrethroid-resistant mosquitoes caused by kdr mutation-induced sodium channel gating modifications.

The potassium channel auxiliary subunit Kvβ2 (Kcnab2) regulates Kv1 channels and dopamine neuron firing.

Ion channel complexes typically consist of both pore-forming subunits and auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here, we use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvβ2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore-forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents toward more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvβ2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvβ2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvβ2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvβ2 subunits and Kv1 complexes in regulating dopamine neuron activity.

Biophysical characteristics of TRIC-A and TRIC-B channels and their regulation of RYR2

Trimeric intracellular cation channels (TRIC-A and TRIC-B), found in the sarco/endoplasmic reticulum (SR/ER) and nuclear membranes, are thought to provide countercurrents to balance Ca2+-movements across the SR, but there is also evidence that they physically interact with ryanodine receptors (RYR). We therefore investigated if TRIC channels could modulate the single-channel function of RYR2 after incorporation of vesicles isolated from HEK293 cells expressing TRIC-A or TRIC-B with RYR2 into artificial membranes under voltage clamp. We also examined the gating and conductance properties of TRIC channels. Co-expression of RYR2 with either TRIC-A or TRIC-B significantly altered the gating behavior of RYR2; however, co-expression with TRIC-A was particularly effective at potentiating the activating effects of cytosolic Ca2+. Fusing membrane vesicles containing TRIC-A or TRIC-B together with RYR2 into bilayers produced large currents of rapidly gating current fluctuations of multiple amplitudes. In 740 cytosolic/210 luminal mM KCl gradient, current-voltage relationships of macroscopic currents revealed average reversal potentials (Erev) of -13.67 ± 9.02 (n = 7), -2.11 ± 3.84 (n = 11), and 13.19 ± 3.23 (n = 13, **, P = 0.0025) from vesicles from RYR2 only, RyR2 + TRIC-A, or RyR2 + TRIC-B cells, respectively. Thus, with the incorporation of TRIC channels, the Erevs depart further from the calculated Erev for ideally selective cation channels than occurs when vesicles from RYR2-only cells are incorporated, suggesting that TRIC channels are permeable to both K+ and Cl-. In conclusion, our results indicate that both TRIC-A and TRIC-B regulate the gating of RYR2, but that TRIC-A has greater capacity to stimulate the RYR2 opening. The results also suggest that TRIC channels may be relatively nonselective ion channels being permeable to both cations and anions. This property would enable TRIC channels to be versatile providers of counter-ion current throughout the SR of many cell types.

Negative cross-resistance of a pyrethroid-resistant Drosophila mutant to Phryma leptostachya-derived haedoxan A.

Voltage-gated sodium channels are the primary target of pyrethroid insecticides. Mutations in sodium channel confer knockdown resistance (kdr) to pyrethroids in various arthropod pests. Haedoxan A (HA) is the major insecticidal component from Phryma leptostachya. It has been shown that HA alters electrical responses at the Drosophila neuromuscular junction and modifies the gating properties of cockroach sodium channels expressed in Xenopus oocytes. However, whether sodium channel mutations that confer pyrethroid resistance also affect the action of HA is unknown. In this study, we conducted bioassays using HA and permethrin in two Drosophila melanogaster strains: w1118 , an insecticide-susceptible strain, and parats1 , a pyrethroid-resistant strain due to a I265N mutation in the sodium channel, and identified a new case of negative cross-resistance (NCR) between permethrin and HA. Both parats1 larvae and adults were more resistant to permethrin, as expected. However, both parats1 larvae and adults were more sensitive to HA compared to w1118 . We confirmed that the I265N mutation reduced the sensitivity to permethrin of a Drosophila sodium channel variant, DmNav 22, expressed in Xenopus oocytes. Interestingly, the I265N mutation also abolished the effect of HA on sodium channels. Further characterization showed that I265 on the sodium channels is critical for the action of both pyrethroids and HA on sodium channels, pointing to an overlapping mode of action between pyrethroids and HA on the sodium channel. Overall, our results suggest an I265N-independnt mechanism(s) in parats1 flies that is responsible for the NCR between permethrin and HA at the whole insect level.

Peripheral Inflammation Results in Increased Excitability of Capsaicin-Insensitive Nociceptive DRG Neurons Mediated by Upregulation of ASICs and Voltage-Gated Ion Channels.

Previously, we have characterized the capsaicin-insensitive low pH-sensitive (caps−lpH+) subtype of small-sized nociceptive dorsal root ganglion (DRG) neurons that express acid-sensing ion channels, T-type Ca2+ channels, and have isolectin B4-negative phenotype. These neurons demonstrated increased excitability in a model of long-term diabetes, contributing to chronic pain sensation. Here we studied changes in the excitability of the caps−lpH+ neurons and underlying changes in the functional expression and gating properties of ion channels under complete Freund's adjuvant (CFA)-induced peripheral inflammation. We have found that, under these pathological conditions, the functional expression of the acid-sensing ion channels (ASICs) and voltage-gated Na+ channels, was increased. In addition, T-type Ca2+ current was significantly increased in the neurons at the membrane potentials close to its resting value. Altogether, the observed changes in the channel functioning shifted a pH level evoking an action potential (AP) toward its physiological value and led to an increase of evoked and spontaneous excitability of the caps−lpH+ neurons that may contribute to hyperalgesia and chronic inflammatory pain.

Atypical antipsychotic olanzapine inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

Olanzapine, an FDA-approved atypical antipsychotic, is widely used to treat schizophrenia and bipolar disorder. In this study, the inhibitory effect of olanzapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells was investigated. Electrophysiological recordings were performed in freshly isolated coronary arterial smooth muscle cells. Olanzapine inhibited the Kv channels in a concentration-dependent manner with an IC50 value of 7.76 ± 1.80µM and a Hill coefficient of 0.82 ± 0.09. Although olanzapine did not change the steady-state activation curve, it shifted the inactivation curve to a more negative potential, suggesting that it inhibited Kv currents by affecting the voltage sensor of the Kv channel. Application of 1 or 2Hz train pulses did not affect the olanzapine-induced inhibition of Kv channels, suggesting that its effect on Kv channels occurs in a use (state)-independent manner. Pretreatment with DPO-1 (Kv1.5 subtype inhibitor) reduced the olanzapine-induced inhibition of Kv currents. In addition, pretreatment with guangxitoxin (Kv2.1 subtype inhibitor) and linopirdine (Kv7 subtype inhibitor) partially decreased the degree of Kv current inhibition. Olanzapine induced membrane depolarization. From these results, we suggest that olanzapine inhibits the Kv channels in a concentration-dependent, but state-independent, manner by affecting the gating properties of Kv channels. The primary Kv channel target of olanzapine is the Kv1.5 subtype.

Hyperexcitability of Nucleus Tractus Solitarii Neurons in the Streptozotocin‐Induced Model of Alzheimer's Disease

Besides the stereotypical problems of memory and cognition in patients with Alzheimer's Disease (AD), another highly prevalent symptom is of respiratory origin. We have repeatedly shown blunted respiratory responses to hypoxia in the streptozotocin (STZ) -induced AD model, which is similar to sleep disordered breathing found in nearly 80% of AD patients. Our current study focuses on the electrophysiological changes within the nucleus tractus solitarii (nTS), a brainstem area known to control respiratory function. Using male Sprague-Dawley rats (n = 15 rats, 240±8 g), we induced AD symptoms with microinjections of 2 mg/kg Streptozotocin (STZ) into the lateral cerebral ventricles. Control animals received 0.9 mM citrate buffer as vehicle control (CTL). The disease model was verified using the Morris water maze to test for memory dysfunction. We recorded nTS neuronal responses via whole cell patch clamp. Action potential (AP) discharge upon current-stimulation was higher in STZ-AD when compared to control. Also, spontaneous spike activity (no stimulation) was elevated. These data corroborate earlier results using a higher (2x1.5 mg) STZ dosage. Spontaneous post-synaptic currents in the absence of stimulation resemble nTS network activity. The number of spontaneous events in nTS neurons of the STZ-AD group was surprisingly similar to CTL in the current study. On the other hand, network activity mirrors the hyperexcitability seen from our current injection when using the higher STZ dosage. While there was no appreciable change in resting membrane potential, AP threshold of STZ-AD nTS cells shifted towards a more negative membrane potential, overall contributing to increased excitability in these cells. AP phase plane plots in STZ-AD had a significantly smaller overshoot with a reduced velocity of up and down stroke, possibly indicating altered function of voltage-gated sodium channels. Analysis of transient K+ currents (A-type) revealed a slight shift to higher activation voltages (from -20 mV [CTL] to 0 mV [STZ-AD]) in STZ-AD. This shift favors AP generation and may contribute to a lower spike threshold in STZ-AD. There were no changes in the steady state K+ currents between groups. In summary, nTS cells of the STZ-AD group showed increased excitability that may involve gating properties of voltage-gated sodium channels and A-type potassium channels. The observed neurophysiological changes in the nTS may be part of the neuronal correlate for increased breathing frequency and blunted (ceiling effect) respiratory responses to hypoxia in the STZ-AD model.

Structural determinants of voltage-gating properties in calcium channels.

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel CaV1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.

Reduced current density, partially rescued by mexiletine, and depolarizing shift in activation of SCN5A W374G channels as a cause of severe form of Brugada syndrome.

Background SCN5A‐related Brugada syndrome (BrS) can be caused by multiple mechanisms including trafficking defects and altered channel gating properties. Most SCN5A mutations at pore region cause trafficking defects, and some of them can be rescued by mexiletine (MEX).ObjectiveWe recently encountered symptomatic siblings with BrS and sought to identify a responsible mutation and reveal its biophysical defects.MethodsTarget panel sequencing was performed. Wild‐type (WT) or identified mutant SCN5A was transfected into tsA201 cells. After incubation of transfected cells with or without 0.1 mM MEX for 24–36 hr, whole‐cell sodium currents (INa) were recorded using patch‐clamp techniques.ResultsThe proband was 29‐year‐old male who experienced cardiopulmonary arrest. Later, his 36‐year‐old sister, who had been suffering from recurrent episodes of syncope since 12 years, was diagnosed with BrS. An SCN5A W374G mutation, located at pore region of domain 1 (D1 pore), was identified in both. The peak density of W374G‐INa was markedly reduced (WT: 521 ± 38 pA/pF, W374G: 60 ± 10 pA/pF, p < .01), and steady‐state activation (SSA) was shifted to depolarizing potentials compared with WT‐INa (V1/2‐WT: −39.1 ± 0.8 mV, W374G: −30.9 ± 1.1 mV, p < .01). Incubation of W374G‐transfected cells with MEX (W374G‐MEX) increased INa density, but it was still reduced compared with WT‐INa (W374G‐MEX: 174 ± 19 pA/pF, p < .01 versus W374G, p < .01 versus WT). The SSA of W374G‐MEX‐INa was comparable to W374G‐INa (V1/2‐W374G‐MEX: −31.6 ± 0.7 mV, P = NS).ConclusionsReduced current density, possibly due to a trafficking defect, and depolarizing shift in activation of SCN5A W374G are underlying biophysical defects in this severe form of BrS. Trafficking defects of SCN5A mutations at D1 pore may be commonly rescued by MEX.

Physiological Functions, Biophysical Properties, and Regulation of KCNQ1 (KV7.1) Potassium Channels.

KCNQ1 (KV7.1) K+ channels are expressed in multiple tissues, including the heart, pancreas, colon, and inner ear. The gene encoding the KCNQ1 protein was discovered by a positional cloning effort to determine the genetic basis of long QT syndrome, an inherited ventricular arrhythmia that can cause sudden death. Mutations in KCNQ1 can also cause other types of arrhythmia (i.e., short QT syndrome, atrial fibrillation) and the gene may also have a role in diabetes and certain cancers. KCNQ1 α-subunits can partner with accessory β-subunits (KCNE1-KCNE5) to form K+-selective channels that have divergent biophysical properties. In the heart, KCNQ1 α-subunits coassemble with KCNE1 β-subunits to form channels that conduct IKs, a very slowly activating delayed rectifier K+ current. KV7.1 channels are highly regulated by PIP2, calmodulin, and phosphorylation, and rich pharmacology includes blockers and gating modulators. Recent biophysical studies and a cryo-EM structure of the KCNQ1-calmodulin complex have provided new insights into KV7.1 channel function, and how interactions between KCNQ1 and KCNE subunits alter the gating properties of heteromultimeric channels.

Methadone Suppresses Neuronal Function and Maturation in Human Cortical Organoids.

Accumulating evidence has suggested that prenatal exposure to methadone causes multiple adverse effects on human brain development. Methadone not only suppresses fetal neurobehavior and alters neural maturation, but also leads to long-term neurological impairment. Due to logistical and ethical issues of accessing human fetal tissue, the effect of methadone on brain development and its underlying mechanisms have not been investigated adequately and are therefore not fully understood. Here, we use human cortical organoids which resemble fetal brain development to examine the effect of methadone on neuronal function and maturation during early development. During development, cortical organoids that are exposed to clinically relevant concentrations of methadone exhibited suppressed maturation of neuronal function. For example, organoids developed from 12th week till 24th week have an about 7-fold increase in AP firing frequency, but only half and a third of this increase was found in organoids exposed to 1 and 10 μM methadone, respectively. We further demonstrated substantial increases in INa (4.5-fold) and IKD (10.8-fold), and continued shifts of Na+ channel activation and inactivation during normal organoid development. Methadone-induced suppression of neuronal function was attributed to the attenuated increase in the densities of INa and IKD and the reduced shift of Na+ channel gating properties. Since normal neuronal electrophysiology and ion channel function are critical for regulating brain development, we believe that the effect of prolonged methadone exposure contributes to the delayed maturation, development fetal brain and potentially for longer term neurologic deficits.

Intrinsic plasticity of cerebellar stellate cells is mediated by NMDA receptor regulation of voltage-gated Na+ channels.

We show that NMDA receptors (NMDARs) elicit a long-term increase in the firing rates of inhibitory stellate cells of the cerebellum NMDARs induce intrinsic plasticity through a Ca2+ - and CaMKII-dependent pathway that drives shifts in the activation and inactivation properties of voltage-gated Na+ (Nav ) channels An identical Ca2+ - and CaMKII-dependent signalling pathway is triggered during whole-cell recording which lowers the action potential threshold by causing a hyperpolarizing shift in the gating properties of Nav channels. Our findings open the more general possibility that NMDAR-mediated intrinsic plasticity found in other cerebellar neurons may involve similar shifts in Nav channel gating. Memory storage in the mammalian brain is mediated not only by long-lasting changes in the efficacy of neurotransmitter receptors but also by long-term modifications to the activity of voltage-gated ion channels. Activity-dependent plasticity of voltage-gated ion channels, or intrinsic plasticity, is found throughout the brain in virtually all neuronal types, including principal cells and interneurons. Although intrinsic plasticity has been identified in neurons of the cerebellum, it has yet to be studied in inhibitory cerebellar stellate cells of the molecular layer which regulate activity outflow from the cerebellar cortex by feedforward inhibition onto Purkinje cells. The study of intrinsic plasticity in stellate cells has been particularly challenging as membrane patch breakthrough in electrophysiology experiments unintentionally triggers changes in spontaneous firing rates. Using cell-attached patch recordings to avoid disruption, we show that activation of extrasynaptic N-methyl-d-aspartate receptors (NMDARs) elicits a long-term increase in the firing properties of stellate cells by stimulating a rise in cytosolic Ca2+ and activation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). An identical signalling pathway is triggered during whole-cell recording which lowers the action potential threshold by causing a hyperpolarizing shift in the gating properties of voltage-gated sodium (Nav ) channels. Together, our findings identify an unappreciated role of Nav channel-dependent intrinsic plasticity in cerebellar stellate cells which, in concert with non-canonical NMDAR signalling, provides the cerebellum with an unconventional mechanism to fine-tune motor behaviour.