Conductance Potassium Channels Research Articles

Mitochondrial Potassium Channels as Druggable Targets.

Mitochondrial potassium channels have been described as important factors in cell pro-life and death phenomena. The activation of mitochondrial potassium channels, such as ATP-regulated or calcium-activated large conductance potassium channels, may have cytoprotective effects in cardiac or neuronal tissue. It has also been shown that inhibition of the mitochondrial Kv1.3 channel may lead to cancer cell death. Hence, in this paper, we examine the concept of the druggability of mitochondrial potassium channels. To what extent are mitochondrial potassium channels an important, novel, and promising drug target in various organs and tissues? The druggability of mitochondrial potassium channels will be discussed within the context of channel molecular identity, the specificity of potassium channel openers and inhibitors, and the unique regulatory properties of mitochondrial potassium channels. Future prospects of the druggability concept of mitochondrial potassium channels will be evaluated in this paper.

Peripheral nerve maturation and excitability properties from early childhood: Comparison of motor and sensory nerves

ObjectiveUnderstanding of maturational properties of sensory and motor axons is of central importance for determining the impact of nerve changes in health and in disease in children and young adults. MethodsThis study investigated maturation of sensory axons using axonal excitability parameters of the median nerve in 47 children, adolescents and young adults (25 males, 22 females; age range 1–25 years) and compared them to concurrent motor studies. ResultsThe overall pattern of sensory maturation was similar to motor maturation demonstrating prolongation of the strength duration time constant (P < 0.001), reduction of hyperpolarising threshold electrotonus (P = 0.002), prolongation of accommodation half-time (P = 0.005), reduction in hyperpolarising current-threshold slope (P = 0.03), and a shift to the right of the refractory cycle curve (P < 0.001), reflecting changes in passive membrane properties and fast potassium channel conductances. Sensory axons, however, had a greater increase in strength duration time constant and more attenuated changes in depolarising threshold electrotonus and current-threshold parameters, attributable to a more depolarised resting membrane potential evident from early childhood and maintained in adults. Peak amplitude was established early in sensory axons whereas motor amplitude increased with age (P < 0.001), reflecting non-axonal motor unit changes. ConclusionsMaturational trajectories of sensory and motor axons were broadly parallel in children and young adults, but sensory-motor differences were initiated early in maturation. SignificanceIdentifying the evolution of biophysical changes within and between sensory and motor axons through childhood and adolescence is fundamental to understanding developmental physiology and interpreting disease-related changes in immature nerves.

Positive Modulation of SK Channel Impedes Neuron-Specific Cytoskeletal Organization and Maturation

N-methyl-D-aspartate receptor (NMDAR) modulates the structural plasticity of dendritic spines by impacting cytoskeletal organization and kinase signaling. In the developing nervous system, activation of NMDAR is pertinent for neuronal migration, neurite differentiation, and cellular organization. Given that small conductance potassium channels (SK2/3) repress NMDAR ionotropic signaling, this study highlights the impact of neonatal SK channel potentiation on adult cortical and hippocampal organization. Neonatal SK channel potentiation was performed by one injection of SK2/3 agonist (CyPPA) into the pallium of mice on postnatal day 2 (P2). When the animals reached adulthood (P55), the hippocampus and cortex were examined to assess neuronal maturation, lamination, and the distribution of synaptic cytoskeletal proteins. Immunodetection of neuronal markers in the brain of P2-treated P55 mice revealed the presence of immature neurons in the upper cortical layers (layers II–IV) and CA1 (hippocampus). Also, layer-dependent cortical-cell density was attenuated due to the ectopic localization of mature (NeuN⁺) and immature (Doublecortin⁺ [DCX⁺]) neurons in cortical layers II–IV. Similarly, the decreased count of NeuN⁺ neurons in the CA1 is accompanied by an increase in the number of immature DCX⁺ neurons. Ectopic localization of neurons in the upper cortex and CA1 caused the dramatic expression of neuron-specific cytoskeletal proteins. In line with this, structural deformity of neuronal projections and the loss of postsynaptic densities suggests that postsynaptic integrity is compromised in the SK2/3⁺ brain. From these results, we deduced that SK channel activity in the developing brain likely impacts neuronal maturation through its effects on cytoskeletal formation.

PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome

The ubiquitin ligase, Ube3a, plays important roles in brain development and functions, since its deficiency results in Angelman Syndrome (AS) while its over-expression increases the risk for autism. We previously showed that the lack of Ube3a-mediated ubiquitination of the Ca2+-activated small conductance potassium channel, SK2, contributes to impairment of synaptic plasticity and learning in AS mice. Synaptic SK2 levels are also regulated by protein kinase A (PKA), which phosphorylates SK2 in its C-terminal domain, facilitating its endocytosis. Here, we report that PKA activation restores theta burst stimulation (TBS)-induced long-term potentiation (LTP) in hippocampal slices from AS mice by enhancing SK2 internalization. While TBS-induced SK2 endocytosis is facilitated by PKA activation, SK2 recycling to synaptic membranes after TBS is inhibited by Ube3a. Molecular and cellular studies confirmed that phosphorylation of SK2 in the C-terminal domain increases its ubiquitination and endocytosis. Finally, PKA activation increases SK2 phosphorylation and ubiquitination in Ube3a-overexpressing mice. Our results indicate that, although both Ube3a-mediated ubiquitination and PKA-induced phosphorylation reduce synaptic SK2 levels, phosphorylation is mainly involved in TBS-induced endocytosis, while ubiquitination predominantly inhibits SK2 recycling. Understanding the complex interactions between PKA and Ube3a in the regulation of SK2 synaptic levels might provide new platforms for developing treatments for AS and various forms of autism.

Age‐Associated Changes in Cerebrovascular Function and BK Channel Function are Sex‐Specific

Large conductance potassium (BK) channels are known to regulate the myogenic reactivity, yet, it is not clear if aging alters the myogenic reactivity via the BK channel in males and females. Thus we hypothesize that age‐associated changes in BK channel subunits modulate the myogenic reactivity in a sex‐specific manner. We used vascular reactivity, patch‐clamp, and biochemical methods to measure the myogenic reactivity, BK channel function, and expression, respectively, in cerebral vessels of adult and aged male and female Sprague Dawley rats. The results suggest that aging and ovariectomy (OVX) exaggerated the myogenic reactivity of middle cerebral arteries (MCA) in females but attenuated it in males. Aging induced outward eutrophic remodeling in females but inward hypertrophic remodeling in males. Aging decreased total, Kv, BK channel currents, and spontaneous transient outward currents (STOC) in vascular smooth muscle cells isolated from females, but not in males. Aging increased BKα subunit mRNA and protein content both in males and females. However, aging decreased BKβ1 subunit protein and mRNA levels in females only. In males, BKβ1 mRNA is increased, but protein is decreased. Iberiotoxin‐induced MCA constriction is lower in aged females but higher in aged males. OVX decreased, but chronic supplementation of estradiol (OVX+17βE rats) restored BK channel expression and function. BKβ1 mRNA is decreased in cerebral vessels of OVX rats that is consistent with decreased protein content. However, supplementation with 17βE did not increase mRNA, but protein is not decreased suggesting that post‐translational mechanisms contribute to the preservation of the protein. Overall the results suggest that aging and OVX‐associated downregulation of the BKβ1 expression and function in females exaggerates the myogenic reactivity of MCA. However, age‐associated increase in BK channel function in males attenuates the myogenic reactivity of MCA.Support or Funding InformationThis work was supported by, in part, by a grant from the Intramural Research Support Program from UMMC and AHA Scientist Development Grant 13SDG14000006 (M. R. Pabbidi).

Evaluation of Hindlimb Arteriolar Vasodilation Evoked by Dynamic Muscle Contraction in Goto‐Kakizaki Rats Using in vivo X‐ray Microangiography

Vascular endothelial dysfunction is a well established comorbidity in diabetes mellitus. In peripheral resistance arteries, it has been reported that endothelium‐dependent hyperpolarization (EDH), which is an important factor to induce vasodilation in small arteries, is downregulated in diabetic animals. We hypothesized that downregulation of EDH inhibits exercise‐induced vasodilation in diabetic patients and causes exercise intolerance. To test this hypothesis, the objective of this study was to evaluate peripheral vascular function in response to muscle contraction and the contribution of EDH to vasodilation in anesthetized diabetic rats. Type 2 diabetic Goto‐Kakizaki (GK) rat and healthy Wistar rat were anesthetized by urethane + α‐chloralose, and then mechanically ventilated. Carotid artery and jugular vein were cannulated for monitoring blood pressure and administering pharmaceutical agents. By means of X‐ray angiography, the hindlimb vasculature was visualized by infusing iodine contrast agent via the catheter inserted into the femoral artery. Angiography was performed before and after rhythmic muscle contraction (0.5 s tetanic contraction + 0.5 s relaxation, repeated 30 times) evoked by sciatic nerve stimulation (40 Hz, 0.1 ms duration). Femoral blood flow was simultaneously monitored by a transit‐time ultrasonic flowmeter. To investigate the contribution of EDH to the changes in arterial diameter and vessel recruitment, angiography was repeated after an intravenous administration of small conductance and big conductance potassium channel inhibitors (50 μg/kg apamin+charybdotoxin), which are known to suppress the EDH‐dependent vasodilation.We observed increases in arterial diameter (~100% increase) in most of hindlimb arteries after the muscle contraction except for the superficial artery and the saphenous artery. We also observed recruitment of small arteries (diameter less than 100 μm), which were not visualized at baseline condition. These vasodilative effects on the hindlimb arteries were similar in GK rat compared to Wistar rat. Increase in femoral blood flow at peak and decrease in vascular resistance at the nadir were also similar between the groups. Inhibition of potassium channels did not affect baseline diameter in arteries and the changes in vasodilation after muscle contraction in either Wistar or GK rats. In conclusion, effects of acute muscle contraction on the changes in arterial diameter and recruitment were preserved in the GK rat. EDH may not contribute to induce the peak response in arterial diameter after muscle contraction.Support or Funding InformationThis work was supported by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan [18K10882].

State-dependent Photocrosslinking at the BK Channel Intersubunit Interface

BK channels are high conductance potassium channels synergistically activated by voltage and Ca2+. Their molecular architecture consists on homotetramers of α-subunits containing a membrane-spanning domain and a cytosolic structure including two RCK-like domains (RCK1 and RCK2). Each RCK contains distinct high affinity Ca2+-binding sites that have been proposed to mediate activation of the BK channel by Ca2+ (Horrigan and Aldrich, 2002; Sweet and Cox., 2008). The binding site in RCK2, known as the Ca2+-bowl, is located very close to the intersubunit interface in the gating-ring. The cryo-EM structure of the full-length BK channel from Aplysia revealed that Ca2+ in this site is additionally coordinated by the side chain of residue N438 from the RCK1 region of the adjacent subunit (human N449; Tao et al., 2017). Functional data with the BKα-N449A mutant from Li et al., 2018; Vouga and Rothberg, 2017, highlight the importance of this residue in the Ca2+ activation mechanism. To further study the role of N449 in BK channel activation by Ca2+ and voltage we took advantage of the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine (BzF), which was incorporated at that specific site in the human BK channel. BK-N449BzF constructs showed functional characteristics undistinguishable from wild-type BK channels. UV-induced BzF photocrosslinking led to a state-dependent reduction of the maximal outward currents. Photocrosslinking of closed channels (UV application during hyperpolarizing pulses) rendered faster and complete current decrease. Conversely, when the UV pulse was applied during the depolarization pulses (open channels), the current decay was incomplete and developed more slowly. Biochemical experiments confirmed intersubunit crosslinking. Further experiments with BK channels including mutations to inactivate the Ca2+ bowl are currently underway to unveil specific structural prerequisites for this intersubunit interaction. Overall, these results implicate a movement at this subunit interface in BK channel activation.

Quantized Three-Ion-Channel Neuron Model for Neural Action Potentials

The Hodgkin-Huxley model describes the conduction of the nervous impulse through the axon, whose membrane's electric response can be described employing multiple connected electric circuits containing capacitors, voltage sources, and conductances. These conductances depend on previous depolarizing membrane voltages, which can be identified with a memory resistive element called memristor. Inspired by the recent quantization of the memristor, a simplified Hodgkin-Huxley model including a single ion channel has been studied in the quantum regime. Here, we study the quantization of the complete Hodgkin-Huxley model, accounting for all three ion channels, and introduce a quantum source, together with an output waveguide as the connection to a subsequent neuron. Our system consists of two memristors and one resistor, describing potassium, sodium, and chloride ion channel conductances, respectively, and a capacitor to account for the axon's membrane capacitance. We study the behavior of both ion channel conductivities and the circuit voltage, and we compare the results with those of the single channel, for a given quantum state of the source. It is remarkable that, in opposition to the single-channel model, we are able to reproduce the voltage spike in an adiabatic regime. Arguing that the circuit voltage is a quantum variable, we find a purely quantum-mechanical contribution in the system voltage's second moment. This work represents a complete study of the Hodgkin-Huxley model in the quantum regime, establishing a recipe for constructing quantum neuron networks with quantum state inputs. This paves the way for advances in hardware-based neuromorphic quantum computing, as well as quantum machine learning, which might be more efficient resource-wise.

Atomistic basis of opening and conduction in mammalian inward rectifier potassium (Kir2.2) channels.

Potassium ion conduction through open potassium channels is essential to control of membrane potentials in all cells. To elucidate the open conformation and hence the mechanism of K+ ion conduction in the classic inward rectifier Kir2.2, we introduced a negative charge (G178D) at the crossing point of the inner helix bundle, the location of ligand-dependent gating. This "forced open" mutation generated channels that were active even in the complete absence of phosphatidylinositol-4,5-bisphosphate (PIP2), an otherwise essential ligand for Kir channel opening. Crystal structures were obtained at a resolution of 3.6 Å without PIP2 bound, or 2.8 Å in complex with PIP2. The latter revealed a slight widening at the helix bundle crossing (HBC) through backbone movement. MD simulations showed that subsequent spontaneous wetting of the pore through the HBC gate region allowed K+ ion movement across the HBC and conduction through the channel. Further simulations reveal atomistic details of the opening process and highlight the role of pore-lining acidic residues in K+ conduction through Kir2 channels.

Quantized Single-Ion-Channel Hodgkin-Huxley Model for Quantum Neurons

The Hodgkin-Huxley model describes the behavior of the cell membrane in neurons, treating each part of it as an electric circuit element, namely capacitors, memristors, and voltage sources. We focus on the activation channel of potassium ions, due to its simplicity, while keeping most of the features displayed by the original model. This reduced version is essentially a classical memristor, a resistor whose resistance depends on the history of electric signals that have crossed it, coupled to a voltage source and a capacitor. Here, we will consider a quantized Hodgkin-Huxley model based on a quantum memristor formalism. We compare the behavior of the membrane voltage and the potassium channel conductance, when the circuit is subjected to AC sources, in both classical and quantum realms. Numerical simulations show an expected adaptation of the considered channel conductance depending on the signal history in all regimes. Remarkably, the computation of higher moments of the voltage manifest purely quantum features related to the circuit zero-point energy. Finally, we study the implementation of the Hodgkin-Huxley quantum memristor as an asymmetric rf SQUID in superconducting circuits. This study may allow the construction of quantum neuron networks inspired in the brain function, as well as the design of neuromorphic quantum architectures for quantum machine learning.

Nucleus accumbens shell small conductance potassium channels underlie adolescent ethanol exposure-induced anxiety

Alcohol use typically begins in adolescence, increasing the likelihood of adult mental disorders such as anxiety. However, the cellular mechanisms underlying the consequences of adolescent alcohol exposure as well as the behavioral consequences remain poorly understood. We examined the effects of adolescent or adult chronic intermittent ethanol (CIE) exposure on intrinsic excitability of striatal medium-sized spiny neurons (MSNs) and anxiety levels. Rats underwent one of the following procedures: (1) light–dark transition (LDT) and open-field (OF) tests to evaluate anxiety levels and general locomotion; (2) whole-cell patch clamp recordings and biocytin labeling to assess excitability of striatal MSNs, as well as morphological properties; and (3) western blot immunostaining to determine small conductance (SK) calcium-activated potassium channel protein levels. Three weeks, but not 2 days, after CIE treatment, adolescent CIE-treated rats showed shorter crossover latency from the light to dark side in the LDT test and higher MSN excitability in the nucleus accumbens shell (NAcS). Furthermore, the amplitude of the medium afterhyperpolarization (mAHP), mediated by SK channels, and SK3 protein levels in the NAcS decreased concomitantly. Finally, increased anxiety levels, increased excitability, and decreased amplitude of mAHP of NAcS MSNs were reversed by SK channel activator 1-EBIO and mimicked by the SK channel blocker apamin. Thus, adolescent ethanol exposure increases adult anxiety-like behavior by downregulating SK channel function and protein expression, which leads to an increase of intrinsic excitability in NAcS MSNs. SK channels in the NAcS may serve as a target to treat adolescent alcohol binge exposure-induced mental disorders, such as anxiety in adulthood.

Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage.

Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

Aging influences cerebrovascular function via BK channel in a sex‐specific manner

The myogenic response of middle cerebral arteries (MCA) protects the brain by altering the diameter in response to lumen pressure to maintain constant cerebral blood flow. Large conductance potassium (BK) channels are known to regulate the myogenic response. However, it is not clear how aging will influence the myogenic response via altering BK channel function in males and females. Here, using vascular reactivity, patch‐clamp and biochemical methods, we found that age‐associated changes in BK channel subunit expression and function alter myogenic response in a sex‐specific manner. Aging exaggerated the myogenic response of MCA of females but did not change the myogenic response in males. Aging abolished differences in myogenic tone of MCA (MT as determined from active and passive diameters) in males and females. Aging, while decreased wall to lumen ratio in females, it increased in males revealing a sex‐specific difference in MCA remodeling. Aging while decreased total K channel and BK channel currents in vascular smooth muscle cells (VSMCs) isolated from female vessels, it did not affect males. Spontaneous transient outward currents (STOC) that represent BK function are also decreased with aging in both males and females. However, the magnitude of the decrease in STOC amplitude and frequency is much greater in aged females than aged males. While aging increased BKα subunit, it decreased β1 subunit protein in cerebral vessels of both males and females. Age‐associated increase in BKα subunit is similar between males and females. However, there is a greater and significant reduction of BKβ1 subunit protein in aged females than aged males. Activation of BKα and β1 subunits either individually or both failed to decrease MT of MCA isolated from aged males and females at physiological lumen pressures. Besides, aging diminished acute estradiol (17βE)‐mediated decrease in MT either alone or combined with BKβ1 agonist in females. However, this combination significantly decreased MT in ovariectomy females that are chronically supplemented with 17βE via increasing BKβ1 protein content. Overall our results suggest that age‐associated changes in cerebral vascular structure and function and that of BK channel subunit expression and function are sex‐specific. Chronic supplementation of 17βE increases BKβ1 protein content, and its activation by agonist combined with acute 17βE attenuates MT in aged females.Support or Funding InformationSupported by AHA SDG (13SDG14000006) and IRSP from UMMC to Mallikarjuna R. Pabbidi.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Voltage-gated calcium channel α 2δ subunits: an assessment of proposed novel roles.

Voltage-gated calcium (CaV) channels are associated with β and α2δ auxiliary subunits. This review will concentrate on the function of the α2δ protein family, which has four members. The canonical role for α2δ subunits is to convey a variety of properties on the CaV1 and CaV2 channels, increasing the density of these channels in the plasma membrane and also enhancing their function. More recently, a diverse spectrum of non-canonical interactions for α2δ proteins has been proposed, some of which involve competition with calcium channels for α2δ or increase α2δ trafficking and others which mediate roles completely unrelated to their calcium channel function. The novel roles for α2δ proteins which will be discussed here include association with low-density lipoprotein receptor-related protein 1 (LRP1), thrombospondins, α-neurexins, prion proteins, large conductance (big) potassium (BK) channels, and N-methyl-d-aspartate (NMDA) receptors.

Axonal sodium and potassium conductance density determines spiking dynamical properties of regular- and fast-spiking neurons

Compared with regular-spiking (RS) neurons, the action potentials (APs) of fast-spiking (FS) neurons are characterized by narrow spike durations and unusually fast decay dynamics of dV/dt process. However, quantitative measure of accurate sodium and potassium densities accounting for spike shape in the axon initial segment (AIS) where action potentials are initiated in both RS and FS cells is not well explored. Here, axonal recordings showed that the averaged axonal spike half-height duration of RS cells is significantly larger (Matlab anova1, $$p < 0.001$$ ) than that of FS cells. The averaged dV/dt ratio of RS cells is significantly lower (Matlab anova1, $$p < 0.001$$ ) than that of FS cells. We have reproduced axonal APs by cortical Hodgkin–Huxley axonal models constrained by a set of experimentally observed properties of RS and FS cells. The model predicts that sodium channel conductance $${g}_{\mathrm{Na}}$$ in RS AIS is in a range of 1000–4000 pS/ $$\upmu \text {m}^{2}$$ and potassium channel conductance $${g}_{\mathrm{K}}$$ is in a range of 30–200 pS/ $$\upmu \text {m}^{2}$$ in order to produce energy-efficient RS action potentials with major properties matching experimental observations of pyramidal cells. The model predicts that $${g}_{\mathrm{Na}}$$ is within 1000–2500 pS/ $$\upmu \text {m}^{2}$$ and $${g}_{\mathrm{K}}$$ ranges within 300–1000 pS/ $$\upmu \text {m}^{2}$$ for AIS of FS cells. The AP duration and efficiency are nonlinearly regulated by the ratio of $${g}_{\mathrm{Na}}/{g}_{\mathrm{K}}$$ . We performed patch-clamp recordings on both RS and FS cortical axons and observed that $${g}_{\mathrm{Na}}$$ was approximately $$1620.4\pm 770\,\hbox {pS}/\upmu \text {m}^{2}$$ for RS AIS and $$803.7\pm 351.5\,\hbox {pS}/\upmu \text {m}^{2}$$ for FS AIS. The $${g}_{\mathrm{K}}$$ was $$189.6\pm 75.8\,\hbox {pS}/\upmu \text {m}^{2}$$ for the RS AIS and $$524.6\pm 281.6\,\hbox {pS}/\upmu \text {m}^{2}$$ for the FS AIS. Partial drug-mediated inhibition of sodium or potassium channels significantly decreases or enlarges the AP duration and dV/dt ratio of both RS and FS cells, respectively, suggesting that sodium and potassium conductance density in cortical axons may be critical in determining the dynamical features of AP profiles in FS and RS cells.

Oxidation of cysteine 117 stimulates constitutive activation of the type Iα cGMP-dependent protein kinase

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2 In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.

Gain control with A-type potassium current: IA as a switch between divisive and subtractive inhibition.

Neurons process and convey information by transforming barrages of synaptic inputs into spiking activity. Synaptic inhibition typically suppresses the output firing activity of a neuron, and is commonly classified as having a subtractive or divisive effect on a neuron’s output firing activity. Subtractive inhibition can narrow the range of inputs that evoke spiking activity by eliminating responses to non-preferred inputs. Divisive inhibition is a form of gain control: it modifies firing rates while preserving the range of inputs that evoke firing activity. Since these two “modes” of inhibition have distinct impacts on neural coding, it is important to understand the biophysical mechanisms that distinguish these response profiles. In this study, we use simulations and mathematical analysis of a neuron model to find the specific conditions (parameter sets) for which inhibitory inputs have subtractive or divisive effects. Significantly, we identify a novel role for the A-type Potassium current (IA). In our model, this fast-activating, slowly-inactivating outward current acts as a switch between subtractive and divisive inhibition. In particular, if IA is strong (large maximal conductance) and fast (activates on a time-scale similar to spike initiation), then inhibition has a subtractive effect on neural firing. In contrast, if IA is weak or insufficiently fast-activating, then inhibition has a divisive effect on neural firing. We explain these findings using dynamical systems methods (plane analysis and fast-slow dissection) to define how a spike threshold condition depends on synaptic inputs and IA. Our findings suggest that neurons can “self-regulate” the gain control effects of inhibition via combinations of synaptic plasticity and/or modulation of the conductance and kinetics of A-type Potassium channels. This novel role for IA would add flexibility to neurons and networks, and may relate to recent observations of divisive inhibitory effects on neurons in the nucleus of the solitary tract.

Chronic Fractalkine Administration Exerts Durable Effects to Improve Glucose Tolerance with Specific Effects to Pancreatic Endocrine Function

Previously we reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and beta cell function. In this study, we examined whether chronic administration of FKN can exert durable effects to improve glucose tolerance in obese rodent models and performed a series of comprehensive mechanistic studies. Chronic administration of a long acting form of FKN (FKN-Fc fusion protein) into obese and insulin resistant mice improved glucose tolerance with improved beta cell function. Unexpectedly, FKN-Fc administration also led to decreased plasma glucagon levels. At the molecular level, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism in both alpha and beta cells. This led to enhanced glucose-stimulated action potential (AP) firing in beta cells, while it decreased alpha cell AP amplitude. Consistent with these results, FKN-Fc treatment increased GSIS and decreased glucagon secretion in beta and alpha cells, respectively. In addition to these effects in islet cells, chronic FKN-Fc treatment also decreased hepatic glucose production. In vitro and ex vivo experiments with normal and obese hepatocytes demonstrated that FKN treatment decreases glucagon-stimulated cAMP levels and CREB phosphorylation in a pertussis toxin-sensitive manner. Taken together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity. Disclosure Y. Lee: None.

Chronic fractalkine administration improves glucose tolerance and pancreatic endocrine function.

We have previously reported that the fractalkine (FKN)/CX3CR1 system represents a novel regulatory mechanism for insulin secretion and β cell function. Here, we demonstrate that chronic administration of a long-acting form of FKN, FKN-Fc, can exert durable effects to improve glucose tolerance with increased glucose-stimulated insulin secretion and decreased β cell apoptosis in obese rodent models. Unexpectedly, chronic FKN-Fc administration also led to decreased α cell glucagon secretion. In islet cells, FKN inhibited ATP-sensitive potassium channel conductance by an ERK-dependent mechanism, which triggered β cell action potential (AP) firing and decreased α cell AP amplitude. This results in increased glucose-stimulated insulin secretion and decreased glucagon secretion. Beyond its islet effects, FKN-Fc also exerted peripheral effects to enhance hepatic insulin sensitivity due to inhibition of glucagon action. In hepatocytes, FKN treatment reduced glucagon-stimulated cAMP production and CREB phosphorylation in a pertussis toxin-sensitive manner. Together, these results raise the possibility of use of FKN-based therapy to improve type 2 diabetes by increasing both insulin secretion and insulin sensitivity.

Lipopolysaccharide stimulates BK channel activity in bladder umbrella cells.

Bladder urothelium plays an active role in response to bacterial infection. There is little known about the electrophysiological activity in urothelial cells in this process. We used a nonenzymatic method to isolate bladder urothelial tissue and to patch clamp umbrella cells in situ. A 200 pS conductance potassium (K+) channel was detected from female C57BL6 mice. Of 58 total patches, 17.2% patches displayed the 200 pS K+ conductance channel. This K+ conductance channel showed Ca2+ sensitivity and voltage dependence. Specific big-conductance potassium channel (BK) inhibitors (paxilline, iberiotoxin) blocked the 200 pS K+ conductance channel activity. RT-PCR and immunoblot confirmed BK channel pore-forming α-subunit (BK-α) mRNA and protein in urothelium. Immunohistochemistry also showed the BK-α located in urothelium. The above data provided evidence that the 200 pS K+ conductance channel was a BK channel. Lipopolysaccharide (LPS), a component of uropathogenic Escherichia coli, was used to investigate the role of BK channel in the pathogenesis of urinary tract infection. BK channel activity as NPo increased threefold within 30 min of exposure to LPS. mRNAs for LPS receptors (TLR4, CD14, MD-2) were expressed in the urothelium but not in lamina propria or detrusor. Blockade of the receptors by an antagonist (polymyxin B) abrogated LPS's effect on BK channel. The involvement of protein kinase A (PKA) on BK channel activity was demonstrated by applying PKA blockers (H89 and PKI). Both PKA inhibitors abolished the BK channel activity induced by LPS. In conclusion, BK channel was identified in bladder umbrella cells, and its activity was significantly increased by LPS.