Structural basis for human Cav1.2 inhibition by multiple drugs and the neurotoxin calciseptine.

Cav1.2 channels play crucial roles in various neuronal and physiological processes. Here, we present cryo-EM structures of human Cav1.2, both in its apo form and in complex with several drugs, as well as the peptide neurotoxin calciseptine. Most structures, apo or bound to calciseptine, amlodipine, or a combination of amiodarone and sofosbuvir, exhibit a consistent inactivated conformation with a sealed gate, three up voltage-sensing domains (VSDs), and a down VSDII. Calciseptine sits on the shoulder of the pore domain, away from the permeation path. In contrast, when pinaverium bromide, an antispasmodic drug, is inserted into a cavity reminiscent of the IFM-binding site in Nav channels, a series of structural changes occur, including upward movement of VSDII coupled with dilation of the selectivity filter and its surrounding segments in repeat III. Meanwhile, S4-5III merges with S5III to become a single helix, resulting in a widened but still non-conductive intracellular gate.

Mono-allelic KCNB2 variants lead to a neurodevelopmental syndrome caused by altered channel inactivation.

Ion channels mediate voltage fluxes or action potentials that are central to the functioning of excitable cells such as neurons. The KCNB family of voltage-gated potassium channels (Kv) consists of two members (KCNB1 and KCNB2) encoded by KCNB1 and KCNB2, respectively. These channels are major contributors to delayed rectifier potassium currents arising from the neuronal soma which modulate overall excitability of neurons. In this study, we identified several mono-allelic pathogenic missense variants in KCNB2, in individuals with a neurodevelopmental syndrome with epilepsy and autism in some individuals. Recurrent dysmorphisms included a broad forehead, synophrys, and digital anomalies. Additionally, we selected three variants where genetic transmission has not been assessed, from two epilepsy studies, for inclusion in our experiments. We characterized channel properties of these variants by expressing them in oocytes of Xenopus laevis and conducting cut-open oocyte voltage clamp electrophysiology. Our datasets indicate no significant change in absolute conductance and conductance-voltage relationships of most disease variants as compared to wild type (WT), when expressed either alone or co-expressed with WT-KCNB2. However, variants c.1141A>G (p.Thr381Ala) and c.641C>T (p.Thr214Met) show complete abrogation of currents when expressed alone with the former exhibiting a left shift in activation midpoint when expressed alone or with WT-KCNB2. The variants we studied, nevertheless, show collective features of increased inactivation shifted to hyperpolarized potentials. We suggest that the effects of the variants on channel inactivation result in hyper-excitability of neurons, which contributes to disease manifestations.

Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Nav1.5.

The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens.

KCNQ Channels Enable Reliable Presynaptic Spiking and Synaptic Transmission at High Frequency.

The presynaptic action potential (AP) is required to drive calcium influx into nerve terminals, resulting in neurotransmitter release. Accordingly, the AP waveform is crucial in determining the timing and strength of synaptic transmission. The calyx of Held nerve terminals of rats of either sex showed minimum changes in AP waveform during high-frequency AP firing. We found that the stability of the calyceal AP waveform requires KCNQ (KV7) K+ channel activation during high-frequency spiking activity. High-frequency presynaptic spikes gradually led to accumulation of KCNQ channels in open states which kept interspike membrane potential sufficiently negative to maintain Na+ channel availability. Blocking KCNQ channels during stimulus trains led to inactivation of presynaptic Na+, and to a lesser extent KV1 channels, thereby reducing the AP amplitude and broadening AP duration. Moreover, blocking KCNQ channels disrupted the stable calcium influx and glutamate release required for reliable synaptic transmission at high frequency. Thus, while KCNQ channels are generally thought to prevent hyperactivity of neurons, we find that in axon terminals these channels function to facilitate reliable high-frequency synaptic signaling needed for sensory information processing.SIGNIFICANCE STATEMENT The presynaptic spike results in calcium influx required for neurotransmitter release. For this reason, the spike waveform is crucial in determining the timing and strength of synaptic transmission. Auditory information is encoded by spikes phase locked to sound frequency at high rates. The calyx of Held nerve terminals in the auditory brainstem show minimum changes in spike waveform during high-frequency spike firing. We found that activation of KCNQ K+ channel builds up during high-frequency firing and its activation helps to maintain a stable spike waveform and reliable synaptic transmission. While KCNQ channels are generally thought to prevent hyperexcitability of neurons, we find that in axon terminals these channels function to facilitate high-frequency synaptic signaling during auditory information processing.

Interneuronal dynamics facilitate the initiation of spike block in cortical microcircuits.

Pyramidal cell spike block is a common occurrence in migraine with aura and epileptic seizures. In both cases, pyramidal cells experience hyperexcitation with rapidly increasing firing rates, major changes in electrochemistry, and ultimately spike block that temporarily terminates neuronal activity. In cortical spreading depression (CSD), spike block propagates as a slowly traveling wave of inactivity through cortical pyramidal cells, which is thought to precede migraine attacks with aura. In seizures, highly synchronized cortical activity can be interspersed with, or terminated by, spike block. While the identifying characteristic of CSD and seizures is the pyramidal cell hyperexcitation, it is currently unknown how the dynamics of the cortical microcircuits and inhibitory interneurons affect the initiation of hyperexcitation and subsequent spike block.We tested the contribution of cortical inhibitory interneurons to the initiation of spike block using a cortical microcircuit model that takes into account changes in ion concentrations that result from neuronal firing. Our results show that interneuronal inhibition provides a wider dynamic range to the circuit and generally improves stability against spike block. Despite these beneficial effects, strong interneuronal firing contributed to rapidly changing extracellular ion concentrations, which facilitated hyperexcitation and led to spike block first in the interneuron and then in the pyramidal cell. In all cases, a loss of interneuronal firing triggered pyramidal cell spike block. However, preventing interneuronal spike block was insufficient to rescue the pyramidal cell from spike block. Our data thus demonstrate that while the role of interneurons in cortical microcircuits is complex, they are critical to the initiation of pyramidal cell spike block. We discuss the implications that localized effects on cortical interneurons have beyond the isolated microcircuit and their contribution to CSD and epileptic seizures.

Impact of intracellular hemin on N-type inactivation of voltage-gated K+ channels.

N-type inactivation of voltage-gated K+ channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary ß subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K+ channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvß1.1, Kvß1.2, and Kvß3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvß1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC50 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary ß subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation.

Resistance to action potential depression of a rat axon terminal in vivo.

The shape of the presynaptic action potential (AP) has a strong impact on neurotransmitter release. Because of the small size of most terminals in the central nervous system, little is known about the regulation of their AP shape during natural firing patterns in vivo. The calyx of Held is a giant axosomatic terminal in the auditory brainstem, whose biophysical properties have been well studied in slices. Here, we made whole-cell recordings from calyceal terminals in newborn rat pups. The calyx showed a characteristic burst firing pattern, which has previously been shown to originate from the cochlea. Surprisingly, even for frequencies over 200 Hz, the AP showed little or no depression. Current injections showed that the rate of rise of the AP depended strongly on its onset potential, and that the membrane potential after the AP (Vafter) was close to the value at which no depression would occur during high-frequency activity. Immunolabeling revealed that Nav1.6 is already present at the calyx shortly after its formation, which was in line with the fast recovery from AP depression that we observed in slice recordings. Our findings thus indicate that fast recovery from depression and an inter-AP membrane potential that minimizes changes on the next AP in vivo, together enable high timing precision of the calyx of Held already shortly after its formation.

Modulation of K+ channel N-type inactivation by sulfhydration through hydrogen sulfide and polysulfides.

Fast N-type inactivation of voltage-gated K+ (Kv) channels is important in fine-tuning of cellular excitability. To serve diverse cellular needs, N-type inactivation is regulated by numerous mechanisms. Here, we address how reactive sulfur species-the gaseous messenger H2S and polysulfides-affect N-type inactivation of the mammalian Kv channels Kv1.4 and Kv3.4. In both channels, the H2S donor NaHS slowed down inactivation with varying potency depending on the "aging" of NaHS solution. Polysulfides were > 1000 times more effective than NaHS with the potency increasing with the number of sulfur atoms (Na2S2 < Na2S3 < Na2S4). In Kv1.4, C13 in the N-terminal ball domain mediates the slowing of inactivation. In recombinant protein exposed to NaHS or Na2S4, a sulfur atom is incorporated at C13 in the protein. In Kv3.4, the N terminus harbors two cysteine residues (C6, C24), and C6 is of primary importance for channel regulation by H2S and polysulfides, with a minor contribution from C24. To fully eliminate the dependence of N-type inactivation on sulfhydration, both cysteine residues must be removed (C6S:C24S). Sulfhydration of a single cysteine residue in the ball-and-chain domain modulates the speed of inactivation but does not remove it entirely. In both Kv1.4 and Kv3.4, polysulfides affected the N-terminal cysteine residues when assayed in the whole-cell configuration; on-cell recordings confirmed that polysulfides also modulate K+ channel inactivation with undisturbed cytosol. These findings have collectively identified reactive sulfur species as potent modulators of N-type inactivation in mammalian Kv channels.

C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation.

Voltage-gated Na+ (NaV) channels are key regulators of myocardial excitability, and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na+ current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts.

Veratridine modifies the gating of human voltage-gated sodium channel Nav1.7.

Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC50) of 18.39 µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC50): 9.53 µM], also in a dose-dependent manner. Veratridine (75 µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64 ± 0.75 mV to -28.14 ± 0.66 mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39 ± 0.39 mV to -73.78 ± 0.5 mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.

Sodium channel biophysics, late sodium current and genetic arrhythmic syndromes.

Arrhythmias arise from breakdown of orderly action potential (AP) activation, propagation and recovery driven by interactive opening and closing of successive voltage-gated ion channels, in which one or more Na+ current components play critical parts. Early peak, Na+ currents (I Na) reflecting channel activation drive the AP upstroke central to cellular activation and its propagation. Sustained late Na+ currents (I Na-L) include contributions from a component with a delayed inactivation timecourse influencing AP duration (APD) and refractoriness, potentially causing pro-arrhythmic phenotypes. The magnitude of I Na-L can be analysed through overlaps or otherwise in the overall voltage dependences of the steady-state properties and kinetics of activation and inactivation of the Na+ conductance. This was useful in analysing repetitive firing associated with paramyotonia congenita in skeletal muscle. Similarly, genetic cardiac Na+ channel abnormalities increasing I Na-L are implicated in triggering phenomena of automaticity, early and delayed afterdepolarisations and arrhythmic substrate. This review illustrates a wide range of situations that may accentuate I Na-L. These include (1) overlaps between steady-state activation and inactivation increasing window current, (2) kinetic deficiencies in Na+ channel inactivation leading to bursting phenomena associated with repetitive channel openings and (3) non-equilibrium gating processes causing channel re-opening due to more rapid recoveries from inactivation. All these biophysical possibilities were identified in a selection of abnormal human SCN5A genotypes. The latter presented as a broad range of clinical arrhythmic phenotypes, for which effective therapeutic intervention would require specific identification and targeting of the diverse electrophysiological abnormalities underlying their increased I Na-L.

Mutagenesis of the NaChBac sodium channel discloses a functional role for a conserved S6 asparagine.

Asparagine is conserved in the S6 transmembrane segments of all voltage-gated sodium, calcium, and TRP channels identified to date. A broad spectrum of channelopathies including cardiac arrhythmias, epilepsy, muscle diseases, and pain disorders is associated with its mutation. To investigate its effects on sodium channel functional properties, we mutated the simple prokaryotic sodium channel NaChBac. Electrophysiological characterization of the N225D mutant reveals that this conservative substitution shifts the voltage-dependence of inactivation by 25 mV to more hyperpolarized potentials. The mutant also displays greater thermostability, as determined by synchrotron radiation circular dichroism spectroscopy studies of purified channels. Based on our analyses of high-resolution structures of NaChBac homologues, we suggest that the side-chain amine group of asparagine 225 forms one or more hydrogen bonds with different channel elements and that these interactions are important for normal channel function. The N225D mutation eliminates these hydrogen bonds and the structural consequences involve an enhanced channel inactivation.

Calcium release-dependent inactivation precedes formation of the tubular system in developing rat cardiac myocytes.

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

Intracellular FGF14 (iFGF14) Is Required for Spontaneous and Evoked Firing in Cerebellar Purkinje Neurons and for Motor Coordination and Balance.

Mutations in FGF14, which encodes intracellular fibroblast growth factor 14 (iFGF14), have been linked to spinocerebellar ataxia (SCA27). In addition, mice lacking Fgf14 (Fgf14(-/-)) exhibit an ataxia phenotype resembling SCA27, accompanied by marked changes in the excitability of cerebellar granule and Purkinje neurons. It is not known, however, whether these phenotypes result from defects in neuronal development or if they reflect a physiological requirement for iFGF14 in the adult cerebellum. Here, we demonstrate that the acute and selective Fgf14-targeted short hairpin RNA (shRNA)-mediated in vivo "knock-down" of iFGF14 in adult Purkinje neurons attenuates spontaneous and evoked action potential firing without measurably affecting the expression or localization of voltage-gated Na(+) (Nav) channels at Purkinje neuron axon initial segments. The selective shRNA-mediated in vivo "knock-down" of iFGF14 in adult Purkinje neurons also impairs motor coordination and balance. Repetitive firing can be restored in Fgf14-targeted shRNA-expressing Purkinje neurons, as well as in Fgf14(-/-) Purkinje neurons, by prior membrane hyperpolarization, suggesting that the iFGF14-mediated regulation of the excitability of mature Purkinje neurons depends on membrane potential. Further experiments revealed that the loss of iFGF14 results in a marked hyperpolarizing shift in the voltage dependence of steady-state inactivation of the Nav currents in adult Purkinje neurons. We also show here that expressing iFGF14 selectively in adult Fgf14(-/-) Purkinje neurons rescues spontaneous firing and improves motor performance. Together, these results demonstrate that iFGF14 is required for spontaneous and evoked action potential firing in adult Purkinje neurons, thereby controlling the output of these cells and the regulation of motor coordination and balance.

Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA).

There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp(67)-Glu(71)-Asp(80) inactivation triad within the channel structure and its bearing on the selectivity filter.

Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species.

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

Alamethicin channel inactivation caused by voltage-driven flux of alamethicin.

We show that voltage alone can inactivate alamethicin channels, which has been previously observed for monazomycin and suzukacillin channels. The voltage required to trigger inactivation is above the potential to form channels, and, like with channel activation, this threshold reduces with increasing peptide concentration and membrane fluidity. Since similar monazomycin channels inactivate via channel break up and translocation, we hypothesized that inactivation of alamethicin channels occurs via the same mechanism. Our data prove this hypothesis to be true through two experiments. First, we show that inactivation of channels at positive voltages when peptides are supplied to only the cis side correlates to new channel activity on the trans side at negative potentials. This result indicates translocation of alamethicin peptides occurs only during voltage-induced inactivation. Second, we measured the ratio of steady-state (with inactivation) to ideal (without inactivation) conductance versus voltage for membranes with equal amounts of alamethicin on both sides and used these values to quantify alamethicin flux. Plotting flux versus steady-state conductance across multiple alamethicin concentrations shows a single linear dependence, signifying that translocated peptides originate from active channels that break up under prolonged voltage. Given the frequent use of alamethicin as model ion channels, these results add important understanding of their kinetic responses when subjected to prolonged, high voltages.

Molecular regulation of the pore component of CRAC channels, Orai1.

Store-operated Ca(2+) entry (SOCE) is a fundamental mechanism ubiquitously employed by cells to elevate intracellular Ca(2+) concentrations ([Ca(2+)]i). Increased intracellular Ca(2+) ions act as a second messenger that can stimulate a variety of downstream signaling pathways affecting proliferation, secretion, differentiation, and death of cells. In immune cells, immune receptor stimulation induces endoplasmic reticulum Ca(2+) store depletion that subsequently activates Ca(2+)-release-activated-Ca(2+) (CRAC) channels, a prototype of store-operated Ca(2+) (SOC) channels. Identification of Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tool to dissect the mechanism of activation and regulation of these channels. In this review, we discuss the recent advances in understanding the regulatory mechanisms and posttranslational modifications that regulate diverse aspects of CRAC channel function.

Regulation of K+ Conductance by a Hydrogen Bond in Kv2.1, Kv2.2, and Kv1.2 Channels.

The slow inactivation of voltage-gated potassium (Kv) channels plays an important role in controlling cellular excitability. Recently, the two hydrogen bonds (H-bonds) formed by W434-D447 and T439-Y445 have been reported to control the slow inactivation in Shaker potassium channels. The four residues are highly conserved among Kv channels. Our objective was to find the roles of the two H-bonds in controlling the slow inactivation of mammalian Kv2.1, Kv2.2, and Kv1.2 channels by point mutation and patch-clamp recording studies. We found that mutations of the residues equivalent to W434 and T439 in Shaker did not change the slow inactivation of the Kv2.1, Kv2.2, and Kv1.2 channels. Surprisingly, breaking of the inter-subunit H-bond formed by W366 and Y376 (Kv2.1 numbering) by various mutations resulted in the complete loss of K+ conductance of the three Kv channels. In conclusion, we found differences in the H-bonds controlling the slow inactivation of the mammalian Kv channels and Shaker channels. Our data provided the first evidence, to our knowledge, that the inter-subunit H-bond formed by W366 and Y376 plays an important role in regulating the K+ conductance of mammalian Kv2.1, Kv2.2, and Kv1.2 channels.

Effects of calcium-binding sites in the S2-S3 loop on human and Nematostella vectensis TRPM2 channel gating processes.

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.