Voltage-gated Potassium Research Articles

A Sharp Rise in Autoimmune Encephalitis in the COVID-19 Era: A Case Series.

Autoimmune encephalitis was very rare prior to the current pandemic. A sharp rise in cases has been observed from March to August of 2022 in Los Angeles. Such an increase, especially with certain types of antibodies, may point toward the possibility of post-infectious autoimmune encephalitis. While review articles on autoimmune encephalitisduring this pandemic have been published, a sharp rise in one geographic area within a short period of time has not been documentedyet. To report an alarming increase in autoimmune encephalitis with mostly positive glutamic acid decarboxylase (GAD) and/or voltage-gated potassium channel (VGKC) antibodies over six months during 2022 in Downtown Los Angeles. This is an observational case series from one neurocritical care practice in Downtown Los Angeles. Autoimmune encephalitis antibody panels were sent to patients with altered mental status or neurologic deficits of unclear etiology from March to August of 2022. Of the 29 patients tested, 12 reports came back positive. Ten had positive GAD and/or VGKC antibodies, one had a positive myelin oligodendrocyte glycoprotein antibody, and one had a positive leucine-richglioma-inactivated 1 protein antibody; a 41% positive rate. This observation has important implications: (1) We may be entering an era of heightened autoimmune encephalitis. (2) These occurrences may be post-infectious in nature at this point of the pandemic. (3) Mostly GAD and VGKC antibodies have been identified (10 of them), which may point toward a new direction of research from a molecular mimicry standpoint. (4) To benefit patients, clinicians need to be aware of such disease manifestations and increase testing; resources must be increased to improve test availability and shorten turnaround time; and treatment, which is expansive, must be made widely available for these potentially reversible diseases.

KCNQ channels open with at least two S4 activation.

Voltage-gated potassium KCNQ2/3 channels mediates the M-current, one of the major potassium currents throughout the central and peripheral nervous systems. KCNQ channels regulate the resting membrane potential, shape action potentials, and impede repetitive neuronal firing. KCNQ channels have six transmembrane segments (S1-S6) that form functional tetramers. The S5-S6 of the four subunits form the pore that is flanked by the four voltage-sensing domains (S1-S4). In response to membrane depolarization the voltage sensor (S4) moves to facilitate potassium conductance. However, the number of voltage-sensor activation required to open the pore remains unknown. Here, we use voltage-clamp fluorometry (VCF) and cysteine accessibility approaches to test for concerted or individual S4 movement required to open KCNQ2/3 channels expressed in Xenopus oocytes. We use the nonconducting E2R mutation in the S2 segment of KCNQ2/3, assumed to restrict voltage sensor movement into activated conformations via electrostatic repulsion with S4 charges, to demonstrate that a concerted motion of all four S4s is not required to open KCNQ channels. VCF shows that homomeric labeled-E2R channels are non-conducting but exhibit voltage-dependent fluorescence signals. Tandem constructs containing labeled-KCNQ and E2R in different subunits (to restrain two S4s in the resting state), render KCNQ2-like currents and a robust voltage-dependent fluorescence signal. In contrast, dimers containing wt-KCNQ and labeled -E2R in the same subunit yield ionic currents with negligible voltage-dependent fluorescence signal. The data suggest that KCNQ channels do not require a concerted movement of all four S4 subunits, but rather that channel opening can occur with at least two S4 movement.

Probing structural topology and dynamics of human KCNE3 membrane protein in lipid bilayers using site-directed spin labeling and electron paramagnetic resonance spectroscopy.

KCNE3 is a single pass membrane spanning protein that modulates the function of the voltage gated potassium ion channel KCNQ1. Genetic mutations in the KCNE3 protein can result in multiple diseases such as long QT syndrome, atrial fibrillation, Mènière's disease, etc. Despite the biological significance, its structural and dynamic properties in a lipid bilayer are not yet fully understood. In this study, the structural topology and dynamic properties of KCNE3 were explored in lipid bilayers utilizing site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. Continuous wave EPR data indicated that the nitroxide spin labeled sidechains of KCNE3 in the transmembrane domain (TMD) are less mobile when compared to that of N and C-termini. Multiple nitroxide spin labeled amino acid sites have been studied using power saturation EPR to determine the membrane depth parameter (φ) of KCNE3 with respect to the POPC/POPG lipid bilayers. These results will be helpful for developing the topology model of KCNE3 in lipid bilayers.

Structure refinement of transmembrane domain of KCNE3 in proteoliposomes using EPR spectroscopy.

KCNE3 is an integral membrane protein that regulates the function of several voltage gated potassium ion channels. A recent solution NMR studies by Sanders lab suggested that the structure of KCNE3 contains a α-helical transmembrane domain with a moderate degree of curvature as an inherent property of the protein. However, detailed structural information of KCNE3 in a lipid bilayer is still not fully understood. In this study, we utilize advanced electron paramagnetic resonance (EPR) technique of double electron electron resonance (DEER) spectroscopy to investigate structural conformation of the transmembrane domain of KCNE3 in proteoliposomes. DEER is a rapidly growing structure biology technique to study structural conformational of membrane protein by measuring distances within the range of 20-80 Å between two spin labeled sites. The DEER distance measurements were obtained for several double spin-labeled sites on the transmembrane domain of KCNE3 in POPC/POPG lipid bilayers. The DEER distance data were used as distances restraints in Xplor-NIH program to refine the structural model of transmembrane domain of KCNE3. This study will provide a guidance to understand structural-functional relationship of transmembrane domain of KCNE3 in lipid bilayer environment.

Protein reconstitution into giant unilamellar vesicles from native nanodiscs.

The reconstitution of purified membrane proteins into lipid bilayers represents a tedious challenge as it relies mainly on empirically optimized purification protocols tailored to the target protein, including laborious detergent selection. The situation is even more complex when the protein's function or structural stability depends on its lipid environment. Using nanodisc-forming amphiphilic polymers helps circumnavigate these problems by allowing the extracted protein to stay in its native lipid environment. Yet, thus far, nanodiscs are of limited use for transport studies due to the problematic protein transfer to vesicles or planar bilayers. Here we exploited the recently introduced glycopolymer Glyco-DIBMA to purify integral membrane proteins such as the voltage-gated potassium ion channel KvAP, the voltage-gated sodium ion channel NavMs, the bacterial translocon complex SecYEG, the eukaryotic serotonin-gated cation channel 5Ht3A, as well as the phospholipid translocating scramblase TMEM16. We used fluorescence correlation spectroscopy to quantify the abundance of the labeled proteins in nanodiscs and optimize protein transfer into giant unilamellar vesicles (GUVs). We thus could achieve protein densities of roughly one copy per diffraction-limited focal area or ∼5400 copies in a GUV with a diameter of ∼20 µm. Such protein density is sufficient for visualizing the reconstituted proteins in vesicular membranes by conventional fluorescence microscopy. The proteins retain functionality, as our GUV influx and efflux studies demonstrated. This project was supported by a grant from the FWF (Austrian Science Fund): P34826.

Structural insights for the modulation of KCNQ1 channel by ML277.

KCNQ1 is a voltage-gated potassium ion channel protein which co-assembles with its accessory regulatory KCNE subunits, KCNE1-5, in order to fulfill its diverse physiological roles in various tissues. In the heart, KCNQ1 helps maintain a normal QT interval in the electrocardiogram, and loss of function mutations can lead to long QT syndrome type 1 (LQT1), seizures, and sudden death. Activators of KCNQ1 may be a promising strategy to oppose the effect of LQT mutations. In this work, we determined the cryo-EM structure of Xenopus KCNQ1 bound to Ca2+/calmodulin with or without the KCNQ1 activator, ML277. Our results showed that a binding site for ML277 lies in the transmembrane domain of the channel which is lined by residues of the S4-S5 linker, and the S5 and S6 helices of two adjacent subunits. MD simulations, along with point mutations, support the binding site location and showed that ML277 binds almost identically in open and closed channels. Comparative structural analyses highlighted important residues for the selectivity of ML277 towards KCNQ1, among other KCNQ subfamily members, and provided an explanation for the limited efficacy of ML277 in the presence of KCNE3. This work paves the way for a better understanding of KCNQ1 regulation by small molecules and may lead to the development of novel and potent activators of KCNQ1, providing a framework for therapeutic intervention targeting KCNQ1 loss of function pathologies.

Kv1 activator mechanism of action in action potential inhibition.

Delayed-rectifier, voltage-gated potassium channels (Kv), such as the Kv1 family, are found in nociceptive dorsal root ganglion (DRG) neurons and represent a potential target for novel analgesics. Activating Kv1 channels in nociceptive neurons may work to decrease neuronal excitability and diminish the sensation of pain. Here, we explored the mechanism by which a Kv1 activator decreases action potential firing in DRG neurons. The small molecules compound A (cmpd-A) and B (cmpd-B) induced a hyperpolarizing shift in the conductance-voltage relationship of a heterologously-expressed Kv1 channel while cmpd-A additionally slowed the deactivation rate in a voltage-independent manner. Cmpd-A, but not cmpd-B, caused a frequency-dependent inhibition of AP firing in rat DRG neurons. We examined the effects of both compounds on Kv1 biophysics and neuronal membrane properties to explain the mechanism of action underlying cmpd-A AP inhibition.

Using rosetta and alphafold to assess Jingzhaotoxin-III binding to hNav1.5 and hKv2.1.

Ion channels are pore-forming transmembrane proteins regulating ion flux across cell membranes and regulators of the ventricular action potential. The human voltage-gated sodium channel 1.5 (hNav1.5) is predominantly expressed in cardiac myocytes and is solely responsible for the ventricular action potential's initial upstroke phase. A series of mutations in hNav1.5 are classified as Long QT Syndrome subtype 3 (LQT3) and are linked to hNav1.5 gain-of-function, resulting in an enhanced inward current prolonging repolarization. Alternatively, improper hNav1.5 inactivation results in a prolonged, late sodium current during the action potential plateau, causing a similar LQT3 gain-of function phenotype. Current drug therapies for hNav1.5-associated arrhythmia involve combinations of beta-adrenergic blockers and hNav1.5-targeting small-molecules such as flecainide. Alternatively, hNav1.5-targeting peptide toxins have the potential as novel therapeutics and investigational tools for ion channel disorders. One such peptide, Jingzhaotoxin-III (JzTx-III) is a 36-residue peptide that has been found to inhibit hNav1.5 and human voltage-gated potassium channel isoform 2.1 (hKv2.1) but does not inhibit neuronal and skeletal hNav channels. Mutational studies of JzTx-III identified unique residues that modulate hNav1.5 and hKv2.1 activity. We hypothesize that prior single-point mutational data will demonstrate differing sets of binding free energies and peptide-channel contacts for hNav1.5 and hKv2.1. In this study, we used AlphaFold and Rosetta to generate models of hNav1.5 and hKv2.1 in the deactivated state, docked JzTx-III to each channel, and assessed in silico binding free energies of wild-type and single-point mutations. This structural and energetic information can then be used as a guideline for multi-point mutational design to predict improved hNav1.5 binding specificity for further investigation of LQTS and pro-arrhythmic ventricular action potentials.

Kv1.3 as a therapeutic target for immunocytoprotection after reperfusion in ischemic stroke.

The voltage-gated potassium channel Kv1.3, which is expressed on activated, disease-associated microglia and memory T cells, constitutes an attractive target for immunocytoprotection after endovascular thrombectomy (EVT). Using young male mice and rats we previously demonstrated that the Kv1.3 blocker PAP-1 when started 12 hours after reperfusion dose-dependently reduces infarction and improves neurological deficit on day-8 after ischemic stroke. However, these proof-of-concept findings are of limited translational value because the majority of strokes occur in patients over 65 and, when considering overall lifetime risk, in females. We therefore tested whether Kv1.3 deletion or delayed pharmacological therapy would be beneficial in females and aged animals. Transient middle cerebral artery occlusion (tMCAO, 60-min) was induced in 16-week and 80-week-old male and female wild-type C57BL/6J and Kv1.3-/- mice. Stroke outcomes were assessed daily with the 14-score tactile and proprioceptive limp-placing test and on day-8 by T2-weighted MRI. Young and old female mice were treated twice daily with 40 mg/kg of the Kv1.3 blocker PAP-1 starting 12 hours after reperfusion. Kv1.3 deletion provided no significant benefit in young females but improved outcomes in young males, old males and old females compared with wild-type controls of the same sex. PAP-1 treatment improved outcomes in both young and old females. In old females, Kv1.3 deletion and PAP-1 treatment significantly reduced microglia activation and T cell infiltration in the infarcted hemisphere. Our preclinical studies using aged and female mice further validate Kv1.3 inhibitors as potential adjunctive treatments for reperfusion therapy in stroke by providing both genetic and pharmacological verification. Using young male rats, we further explored the treatment window for Kv1.3 inhibitors and found that PAP-1 treatment still provided benefit and reduced inflammatory marker expression in the brain when started 72 hours after reperfusion.

Modulation of delayed rectifying K+ currents by silent Kv subunits.

Voltage-gated potassium channels (VGKCs) play fundamental roles in neurobiology; they are a major hub for potential modulation of cell excitability and health. VGKCs are formed by homotetramerization of K+ channels subunits (Kv1-12). Kv2 channels are the major cause of the delayed rectifying K+ currents (IKdr) that tune neuronal firing frequency. Uniquely, Kv2 subunits can co-assemble with members of four other families of Kv (Kv5-6, 8-9). These other Kvs are named silent since they do not form functional channels themselves as homomeric VGKCs. Heteromeric co-assemblies (hetVGKCs) form biophysically distinct channels that modify IKdr and therefore determine neuronal excitability. More than that, the expression of silent subunits suppresses the overall expression of Kv2 subunits, affecting the abundancy of homomeric Kv2 channels and heteromeric channels containing Kv2 subunits. To date, no established mechanism explains how silent Kv subunits interfere with hetVGKCs gating processes or how the mechanism of suppression works. Here, we used Kv6.4 as archetypal silent subunit expressed together with Kv2.1 in HEK cells. Keeping the total amount of Kv2.1 plasmid constant, we found a significant decrease in the magnitude of currents with increasing Kv6.4 dose (ANOVA, p<0.05). To more cleanly assess biophysical differences between homo- and heterotetramers, we generated a Kv2.1-Kv6.4 dimer and compared currents recorded from cells transfected with Kv2.1 plasmid only vs those transfected with dimer plasmid only. We found a significant hyperpolarizing shift in inactivation V50 (−79.6 mV vs −53.5 mV, t-test, p=0.0001), significantly larger activation slope (20.7 vs 14.9, t-test, p=0.0014) and faster activation kinetics (2-way ANOVA, p=0.0354) for the dimer currents compared to Kv2.1-only currents. These results highlight several ways in which silent subunits modify the function of Kv2.1 currents.

Introducing electrostatic stimulation to normal mode analysis.

Normal Mode Analysis (NMA) has proven to be a powerful tool to understand the macromolecular movement of protein complexes. While less precise than an all-atom molecular dynamics simulation, it allows to predict movements on timescales that are not yet accessible to MD simulations on a routine basis. In this work, we build on the previous successful implementation EnCoM to include electrostatic interactions in the NMA analysis. The motivation behind this step is to study the conformational rearrangements of voltage-gated ion channels that are sensitive to the electric field over the membrane. We show here preliminary results on the extension of the inter-residue interactions to include electrostatics and first results on the effect of an electric field on the normal modes of a voltage-gated potassium (Kv) channel.

KCNH2 encodes a nuclear-targeted polypeptide that mediates hERG1 channel gating and expression.

KCNH2 encodes hERG1, the voltage-gated potassium channel that conducts the rapid delayed rectifier potassium current (IKr) in human cardiac tissue. hERG1 is one of the first channels expressed during early cardiac development, and its dysfunction is associated with intrauterine fetal death, sudden infant death syndrome, cardiac arrhythmia, and sudden cardiac death. Here, we identified a novel hERG1 polypeptide (hERG1NP) that is targeted to the nuclei of immature cardiac cells, including hiPSC-CMs and neonatal rat cardiomyocytes. The nuclear hERG1NP immunofluorescent signal is diminished in matured hiPSC-CMs and absent from adult rat cardiomyocytes. Antibodies targeting distinct hERG1 channel epitopes demonstrated that the hERG1NP signal maps to the hERG1 distal C-terminal domain. KCNH2 deletion using CRISPR simultaneously abolished IKr and the hERG1NP signal in hiPSC-CMs. We then identified a putative nuclear localization sequence (NLS) within the distal hERG1 C-terminus, 883-RQRKRKLSFR-892. Interestingly, the distal hERG1 C-terminal domain was targeted almost exclusively to the nuclei when overexpressed HEK293 cells. Conversely, deleting the NLS from the distal peptide abolished nuclear targeting. Similarly, blocking α or β1 karyopherin activity diminished nuclear targeting. Finally, overexpressing the distal hERG1 C-terminus significantly reduced hERG1a current density and slowed deactivation, compared to cells expressing GFP. Deleting the NLS from the distal hERG1 C-terminal domain abolished the effects of the distal C-terminus on hERG1a current amplitude and kinetics. These data identify a developmentally regulated polypeptide encoded by KCNH2, hERGNP, whose presence in the nucleus indirectly modulates hERG1 current magnitude and kinetics.

Further characterization of two oppositely acting, epilepsy-associated mutations at position H310 of Kv1.2 (KCNA2) channels.

KV1.2, encoded by KCNA2, is a prominent voltage-gated potassium channel that controls neuronal excitability. Two KCNA2 mutations (p.H310Y, p.H310R), within the voltage-sensing domain (VSD) have been associated with epileptic encephalopathy and intellectual disability. Last year we showed that H310Y had a double gain-of-function effect, increasing surface trafficking and slowing channel closure by ∼4-fold. Despite producing similar clinical outcomes, H310R showed a loss-of-function effect, abolishing surface trafficking. Here, we further characterize the properties of these mutations.

Competition between Slc7a5 and LMAN2 for modulation of Kv1.2 gating.

The voltage-gated potassium channel Kv1.2 is a prominent ion channel in the CNS, where it regulates electrical activity. Kv1.2 structure and function are well understood, but its regulation by auxiliary proteins has not been widely studied. We identified novel regulators of Kv1.2 by a mass spectrometry approach, including the neutral amino acid transporter Slc7a5, which causes a dramatic hyperpolarizing shift of channel activation. In contrast, LMAN2 is a recently identified candidate regulator that has the opposite effect on gating: large depolarizing voltages are required to activate Kv1.2 channels co-expressed with LMAN2. In this study, we characterized the functional interaction between LMAN2 and Slc7a5 on Kv1.2 gating properties, and identified key structural elements that underlie sensitivity to either regulator. When LMAN2 and Slc7a5 are expressed together, Kv1.2 activation exhibits a bimodal voltage-dependence, suggesting two distinct populations of channels regulated either by LMAN2 or Slc7a5, but not both. Kv1.5 is a close homolog of Kv1.2, but insensitive to both LMAN2 and Slc7a5. Using a Kv1.2:1.5 chimeric approach, we identified the S1 segment of the voltage sensing domain (VSD) as essential for Slc7a5 sensitivity, and the S2-S3 linker of the VSD for LMAN2 sensitivity. By replacing either segment with sequence from Kv1.5, modulation by the corresponding regulator was selectively abolished. Replacement of the S1-S3 segment of Kv1.2 with sequence from Kv1.5 rendered the channel insensitive to both forms of regulation. These results suggest that Slc7a5 and LMAN2 compete for interaction with the Kv1.2 voltage sensor, leading to complex voltage-dependence of channel activity when both regulators are present in the cell. Although the structural basis for this modulation remains uncertain, Kv1.2 clearly exhibits a propensity for modulation over a wide range of voltages.

Mutations affecting electro-mechanical coupling in KCNQ channels alter kinetics and voltage dependence of activation.

The KCNQ voltage-gated potassium channel family serves multiple physiological functions, from regulating heartbeat (by KCNQ1) to controlling neuronal excitability (by KCNQ2-5). In KCNQ1 channels, two steps of voltage sensor activation lead to two functionally distinct open states, the intermediate-open (IO) and activated-open (AO), which define the gating, pharmacology, and physiology of KCNQ1. However, whether all KCNQ channels share the same mechanism is unclear. Here, we provide functional evidence that KCNQ2/3 only conducts at the AO state, but the electro-mechanical (E-M) coupling mechanism is conserved between KCNQ1 and KCNQ2/3. We demonstrate that this conserved E-M coupling can be compromised by select epilepsy-linked mutations in KCNQ2/3 and also can be effectively targeted by the antiepileptic prototype drug retigabine, suggesting E-M coupling as a therapeutic target for epilepsy treatment. Interestingly, KCNQ1 and KCNQ2/3 exhibit distinct kinetics and voltage dependence of activation for the AO state. While these differences may have important implications for the primary physiological roles of KCNQ2/3 (modulation of neuronal excitability) versus KCNQ1 (repolarization of cardiac action potential), it remains unclear where they originate from. Here, we find that analogous mutations in the distal S6 of KCNQ1 and KCNQ2 both affect the AO state E-M coupling and lead to delays in current activation kinetics and right shifts in voltage dependence of activation. KCNQ2 mutations near this location are also found to be associated with epileptic encephalopathy. These results further support a common regulatory mechanism for KCNQ currents via E-M coupling and indicate the physiological relevance of altering AO state activation properties in KCNQ channels.

Kv1.3-dependent immune system activation is regulated by KCNE4.

Kv1.3 is a voltage-gated potassium channel that regulates leukocyte physiology. The association of Kv1.3 with the regulatory subunit KCNE4 fine-tunes the channel's activity. KCNE4 is a transmembrane protein that interacts with Kv1.3 through both of their cytoplasmic C-terminal ends. The protein regulates the membrane trafficking of the channel, affecting the magnitude of the currents and the C-type inactivation. Anomalies in Kv1.3 activity cause several autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, or multiple sclerosis. Therefore, the therapeutic relevance of Kv1.3 postulates KCNE4 as a potential modulator of the immune system physiology. In this study, we investigated the effect of KCNE4 on the activity of Kv1.3 in different leukocyte cell lines. We analyzed the expression of KCNE4 in a model of Jurkat T-lymphocytes and in CY15 dendritic antigen-presenting cells. Dendritic cells exhibit a high expression of the ancillary peptide. Manipulation of KCNE4 abundance affected the Kv1.3-dependent T cell physiology, having an impact on the channel's translocation to the immunological synapse (IS) and IL-2 production. In CY15 cells, activation with LPS increased the Kv1.3/KCNE4 ratio and the current density of the cells. The oligomeric Kv1.3-KCNE4 channel has a variable stoichiometry as demonstrated by single fluorescence bleaching steps. Each tetramer can bind up to four molecules of KCNE4, affecting plasma membrane trafficking and current density of Kv1.3 in a progressive manner. Contrarily, a single KCNE4 subunit was able to enhance the C-type inactivation of the channel. Our results prove that KCNE4 acts as a regulatory element in Kv1.3-dependent immune system physiology. Kv1.3 is differentially modulated by the KCNE4 availability influencing the Kv1.3/KCNE4 architecture of the oligomeric channel and its physiological function. Supported by the Ministerio de Ciencia e Innovación (MICINN/AEI), Spain (PID2020-112647RB-I00 and 10.13039/501100011033) and European Regional Development Fund (FEDER).

Mitochondrial dynamics control of mitoKv1.3 during cell cycle progression.

The voltage-gated potassium channel Kv1.3 plays a crucial role in adipose tissue. Kv1.3 is involved in the development of obesity and diabetes, thus becoming a potential therapeutical target. Kv1.3 locates at the plasma membrane and at mitochondria, controlling cell proliferation and differentiation or apoptosis, respectively. While the plasma membrane targeting is by a COPII-dependent mechanism and driven by several forward-traffic motifs, little is known about the mitochondrial route. In this work, the mitochondrial Kv1.3 interactome was obtained in order to decipher the mitochondrial components that participate in mitoKv1.3 import pathway. We report for the first time that, although Kv1.3 is a multispanning protein with no defined N-terminal presequence, the channel follows an unconventional routing through the TIM23 complex. The targeting is mediated by the information encoded along the channel sequence and regulated by the cytosolic Hsp70/90 chaperon complex. Once in mitochondria, mitoKv1.3 shows a higher affinity for perinuclear populations. In addition, mitoKv1.3 expression is cell cycle-dependent, increasing at the G1/S phase. The development of a knocked-down preadipocyte cell line let us describe that Kv1.3 promotes the preadipocyte proliferation by regulating mitochondrial dynamics. Throughout the cell cycle, there are several fusion-fission events that are key for a correct proliferation. We found that Kv1.3 expression mediates the G1/S phase hyperfusion and, consequently, the hyperpolarization of mitochondria. Our results unravel the unconventional import pathway of Kv1.3 into the inner mitochondrial membrane. Moreover, it sheds light into Kv1.3 control of mitochondrial morphology and dynamics at the G1 phase fine-tuning proliferation. Supported by the Ministerio de Ciencia e Innovación (MICINN/AEI), Spain (PID2020-112647RB-I00 and 10.13039/501100011033) and European Regional Development Fund (FEDER).

Potassium channel openers targeting different sites of the channel.

Voltage-gated ion channels play essential roles in generating and shaping the electrical excitability of nerve and heart cells. Mutations in these ion channels can cause diseases, which can be treated with pharmaceuticals targeting specific ion channels. While many pharmaceuticals cause their effects by blocking the ion-conducting pore to prevent the ion current, only a few exert their therapeutic effect by opening (or activating) the channel. While they see limited use as pharmaceuticals at present, recent research has identified many different types of compounds that can open ion channels. Here, I will present data from three classes of compounds capable of opening voltage-gated potassium (Kv) channels. (1) The first class includes resin acids and polyunsaturated fatty acids. These negatively charged compounds have close interactions with the channel's positively charged voltage sensor, opening the channel by pulling the sensor to an activated position. This interaction occurs at the interface between the phospholipid bilayer and the channel protein. (2) Biaryl sulfonamide acids can access the voltage sensor through the extracellular space, allowing them to activate the Kv1-like Shaker channel. (3) Warfarine-like tautomers activate Kv1.5 channels via close interactions with the linker connecting the voltage sensor and the channel's ion conducting pore domain. Characterizing different binding sites and compounds that use them on different ion channels will hopefully lead to better treatments for diseases caused by altered excitability.

Modulation of Kv1.2 redox-sensitive gating by the transmembrane lectin LMAN2.

The voltage-gated potassium channel Kv1.2 plays a pivotal role in the nervous system, where it regulates membrane excitability and action potential propagation. Compared to other Kv1 channels, Kv1.2 exhibits dynamic voltage-dependent gating with dramatic cell-to-cell variability that is attributed to regulation by unidentified external factors. Recent progress from our lab has identified extrinsic factors that shift the voltage-dependence of Kv1.2 gating. For example, the neutral amino acid transporter Slc7a5 causes a dramatic hyperpolarizing shift of Kv1.2 channel activation. A counteracting effect is that extracellular reducing conditions promote a depolarizing shift in channel activation. However, the underlying mechanism of redox sensitivity has been challenging to identify. Using a mass-spectrometry approach to identify novel Kv1.2 accessory proteins, followed by screening candidates with whole-cell patch clamp electrophysiology, we observed that co-expression with the transmembrane lectin LMAN2 caused a pronounced depolarizing shift of Kv1.2 voltage-dependence. Overexpression of LMAN2 enhanced Kv1.2 sensitivity to extracellular reducing conditions, while shRNA knockdown of endogenous LMAN2 abrogated the redox effects. Using a chimeric approach between Kv1.2 and the LMAN2 insensitive Kv1.5 subunit, we found the S2-S3 linker of the VSD to be essential for sensitivity to both LMAN2 and reducing conditions. Biotinylation experiments revealed that LMAN2 maturation to the plasma membrane is required for its effects on Kv1.2 gating. Furthermore, extracellular application of the membrane impermeable reducing agent TCEP to dorsal root ganglion (DRG) neurons, decelerated the activation kinetics of isolated Kv1.2 currents and increased excitability. In conclusion, we identified a novel accessory protein (the transmembrane Lectin, LMAN2) that alters Kv1.2 gating properties and contributes to redox sensitivity in heterologous cells and DRG neurons.

Functional impact of the interaction between S1 segment and KCNE subunit in the modulation of KCNQ1 channels.

KCNQ1 channel is a voltage-gated potassium channel broadly expressed in the human body, and KCNE proteins are modulatory subunits for the KCNQ1 channel. Among five members of the KCNE subfamily, at least KCNE1 and KCNE3 are considered to modulate the KCNQ1 channel through the voltage sensor domain (VSD), probably by stabilizing its intermediate state. However, the mechanisms of how the KCNE subunits interact and affect the VSD movements are still not fully understood. The recent cryo-EM structure of the KCNQ1-KCNE3 complex shows close interaction between the S1 segment and KCNE3 throughout the transmembrane region. Therefore, we hypothesized that the KCNE subunit controls the VSD equilibrium by interacting with the S1 segment. To test that, we mutated seven amino acid residues on the S1 (F123, F127, F130, L134, I138, L142, and I145) facing the KCNE3 subunit to four different hydrophobic amino acid residues with various sizes for each (“volume scanning”). The KCNE3 modulation depended on the size of introduced amino acid residues at most sites: The more different the size of amino acid residue from the wild-type, the more constitutive openness was weakened. A similar tendency was also observed in KCNE3 mutants: we introduced five or six different amino acid residues to each of the KCNE3 amino acid residues facing the S1 (S57, I61, M65, A69, G73, and I76), and the KCNE3 modulation depended on the size of introduced amino acid residues. Finally, we found two volume swap pairs (KCNQ1 F127A-KCNE3 G73L; KCNQ1 I145F-KCNE3 S57A), which restored the normal function (stabilizing the VSD at the intermediate state) of the KCNQ1-KCNE3 channels. These results indicate that the interaction between the S1 and KCNE3 is tightly optimized to regulate the VSD equilibrium.