Kv Channel Activity Research Articles

Hydrogen sulfide impairs shear stress-induced vasodilation in mouse coronary arteries.

Hydrogen sulfide has emerged as an important endothelium-dependent vasodilator, but its role in shear stress-mediated dilation of coronary arteries is unclear. We examined the role of H2S on shear stress-mediated dilation of isolated mouse coronary arteries. In these vessels, Na2S produced concentration-dependent dilation, which was significantly inhibited by iberiotoxin and by 4-aminopyridine. In addition, BK and Kv currents in mouse coronary smooth muscle cells were directly activated by Na2S, suggesting that H2S produced vasodilation through BK and Kv channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25 dynes/cm(2)) and produced graded dilatory responses, but such effects were diminished in the presence of 100 μM Na2S. Pre-incubation with the cystathionine γ-lyase inhibitor, D,L-propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress-mediated vasodilation. However, in the presence of L-NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress-mediated vasodilation, suggesting an interaction between NO and H2S signaling. Na2S inhibited eNOS activity in cultured mouse aortic endothelial cells and reduced the level of phospho-eNOS(serine 1177). These results suggest that both NO and H2S are important shear stress-mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H2S through inhibition of NO generation.

Testosterone and atrial natriuretic peptide share the same pathway to induce vasorelaxation of human umbilical artery.

We recently observed in human umbilical artery smooth muscle cells that testosterone activates protein kinase G and stimulates large-conductance Ca²⁺ activated (BKCa) and voltage sensitive (KV) potassium channels. In the same work, we also show that atrial natriuretic peptide (ANP), an activator of particulate guanylate cyclase (pGC), stimulates the activity of BKCa and KV channels because of protein kinase G activation. The aim of this work was to prove that the relaxant effects of testosterone are also because of the increase of cGMP because of activation of the pGC. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (ICNG) in human umbilical artery smooth muscle cells expressing the wild-type rat olfactory cyclic nucleotide-gated (CNG) channel. Sodium nitroprusside (10 and 100 μM), ANP (0.1 and 1 μM), or testosterone (0.1, 1, and 10 μM) induced activation of ICNG. This activation induced by testosterone and ANP is bigger than that elicited by sodium nitroprusside. In summary, our study reveals that testosterone and ANP activate the pGC and induce vasorelaxation of human umbilical artery.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca2+-activated K+ channels and inward rectifier K+ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-014-1513-3) contains supplementary material, which is available to authorized users.

The Kv7 potassium channel activator retigabine decreases alcohol consumption in rats

Background: Activation of Kv7 potassium channels may decrease the reactivity of mesolimbic dopaminergic neurons that are implicated in mediating the reinforcing effects of ethanol. Objectives: The objective of this study was to determine whether the administration of the Kv7 potassium channel opener retigabine would decrease ethanol intake in Long Evans rats. Methods: A limited access two-bottle choice model of alcohol (10% solution) consumption was used in this study. A separate group of animals was tested to evaluate the actions of retigabine on sucrose (5% solution) consumption to determine whether this drug might produce non-selective impairment of the ability of rats to drink liquids. Animals were treated with either vehicle or increasing doses (2.5–7.5 mg/kg SC) of retigabine administered over a 3-day period. Results: Compared to vehicle, retigabine at a dose of 7.5 mg/kg produced a reduction in the amount of ethanol consumed. These effects did not occur in association with significant changes in water consumption. A significant time effect was found for the actions of retigabine in sucrose-drinking rats with a trend for an increase in sucrose intake with the highest dose of retigabine administered. Conclusions: These results indicate that the administration of retigabine may produce a decrease in ethanol consumption by rats at doses that do not significantly reduce the drinking of either water or a sucrose solution. These findings are consistent with the hypothesis that activation of Kv7 channels facilitates the reduction of alcohol consumption in the rat.

K+ channels as potential targets for the treatment of gastrointestinal motor disorders

K+ channels play important functional roles in excitable cells, as neurons and muscle cells. The activation or inhibition of K+ channels hyperpolarizes or depolarizes the cell membrane, respectively. These effects determine in the smooth muscle decrease or increase in Ca2+ influx through voltage-gated Ca2+ (CaV1.2) channels and relaxation or contraction, respectively. Recent studies highlight the importance of voltage-dependent type 7 K+ (KV7 or KCNQ) channels in regulating muscle tone and contractility in stomach and colon. KV7 channels, that include 5 subtypes (KV7.1–7.5), are activated at relatively negative potential values, close to those of the resting membrane potential for the smooth muscle cells of some segments of the gastrointestinal tract. Thus, they contribute to set the resting membrane potential and their blockade induces increase in smooth muscle contractility in stomach and colon. In addition, KV7 channel activation produces profound relaxations of gastric and colonic smooth muscle. Therefore, KV7 channel activators could be used to relax the smooth muscle and relieve symptoms in diseases such as functional dyspepsia and irritable bowel syndrome with prevalent diarrhea. The discovery of activators selective for the channel subtypes present in the smooth muscle, mainly KV7.4 and 7.5, would allow avoiding adverse cardiac and nervous system effects. A further step forward would be characterizing putative differences among the KV7 channel subtypes expressed in the various smooth muscles and synthesizing molecules specific for the gastrointestinal smooth muscle.

A novel approach to bronchodilation: combining the Kv7 channel activator retigabine with the β2‐adrenergic agonist formoterol (660.9)

Kv7 potassium channels are expressed in airway smooth muscle cells (ASMCs) and bronchoconstrictor agonists, such as methacholine and histamine, inhibit Kv7 currents. Inhibition of Kv7 channels in rat and human airways is sufficient to induce bronchoconstriction. Long‐acting β2‐adrenergic agonists (LABAs) are commonly used in the treatment of asthma. However, long term use of these drugs may lead to tolerance. Here we provide evidence that the Kv7 channel activator retigabine can oppose agonist‐induced suppression of Kv7 currents and enhance bronchodilation by the LABA formoterol. Dose‐dependent bronchodilation by retigabine and formoterol was measured using rat precision cut lung slices (PCLS). A submaximal dose of formoterol (10nM) induced relaxation of methacholine‐constricted airways, but this relaxation was transient despite continued treatment. The approximate EC50 dose of retigabine (10µM) enhanced formoterol‐induced relaxation and eliminated the apparent desensitization. Similar results were obtained in human PCLS pre‐constricted with histamine, suggesting a beneficial effect of combining a Kv7 channel activator with standard bronchodilator therapy.Grant Funding Source: Supported by Loyola University Chicago

Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1), C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1(+/+) stem cell line do not display the hyperexcitability phenotype. SOD1(A4V/+) ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.

Contribution of Kv1 channel activity to myogenic responses in skeletal muscle arterioles with aging and exercise training (678.7)

The impaired myogenic constriction of skeletal muscle arterioles observed with advancing age may be the result of increased Kv channel activity. Exercise training has been shown to restore myogenic responses. The purpose of this study was to determine the role of Kv1 channel activity in these adaptations in reactivity.Young and old male Fischer 344 rats were randomly assigned to either a sedentary (YSED and OSED) or exercise trained (YET and OET) group. Training consisted of treadmill running (15m/min, 15°incline, 1hr/d, 5d/wk for 10 wks). Soleus muscle first order arterioles were isolated and cannulated for in vitro study. Myogenic responsiveness was determined by exposing arterioles to incremental changes in intraluminal pressure from 0 to 140 cmH20, either in the presence or absence of Kv1 channel blocker correolide.Exercise enhanced myogenic tone in arterioles from both old and young rats and restored myogenic responsiveness in old rats to that of YSED rats. Kv1 channel blockade abolished the training effect in young rats, and resulted in greater constriction at lower pressures in YSED, but had no effect in YET rats. In both OSED and OET rats, Kv1 channel blockade led to increased myogenic constriction; however, the training effects persisted.These results suggest that Kv1 channel activity may play a role in modulating myogenic tone in young and old rats. Increased myogenic constriction after training in young rats may be mediated through a reduction in Kv1 channel activity. However, improved myogenic responsiveness with training in old rats appears to be mediated through a different pathway.Grant Funding Source: Supported by NIH R01HL090937

Kv7 channels as targets for anti-epileptic and psychiatric drug-development

The Kv7 channels, a family of voltage-dependent K+ channels (Kv7.1–Kv7.5), have gained much attention in drug discovery especially because four members are genetically linked to diseases. For disorders of the CNS focus was originally on epilepsy and pain, but it is becoming increasingly evident that Kv7 channels can also be valid targets for psychiatric disorders, such as anxiety and mania. The common denominator is probably neuronal hyperexcitability in different brain areas, which can be successfully attenuated by pharmacological increment of Kv7 channel activity. This perspective attempts to review the current status and challenges for CNS drug discovery based on Kv7 channels as targets for neurological and psychiatric indications with special focus on selectivity and mode-of-actions.

Lipid Affinity to the Voltage-Gated Potassium Channel KvAP

Voltage-gated potassium channels (KV) are formed by a central conducting pore surrounded by four voltage sensor domains. Functional studies have revealed that biophysical properties of lipid molecules in the channels environment can have strong effects on the activity of KV channels. Here, we investigated the influence of different lipids as well as their affinity to KvAP channels. We carried out electrophysiology measurements by fusing vesicles containing purified channels into planar lipid bilayers with varied lipid compositions. We found that KvAP properties are mainly determined by the lipid composition in the vesicles and not the lipids in the bulk bilayer, suggesting that KvAP has a preferential affinity to the lipids it comes in contact with first. It also shows that there is very limited exchange of the annular lipids around the channels. Even with time and at higher temperature, lipids from the bilayer did not mix with those of the vesicles, suggesting a strong channel-lipid interaction or confinement of lipid molecules to a microdomain around the channel. The annular lipids in immediate proximity of the VSD determines the mid-activation point of KvAP. Additionally, a bulk effect from positively charged lipids forming the bilayer affected the slope of the conductance-voltage curves. Here, the energy barriers and thus the kinetic rates of pore opening and entering the inactivated state are affected. This effect could be neutralized by addition of counter charges to the bilayer. Our results suggest that the binding site proposed also for other Kv channels has a similar affinity for different phospho- and non-phospholipids whereas exchange with bulk lipids is very restricted. Apart from the structural information, this has also implications on the experimental design when working with membrane proteins reconstituted in planar lipid bilayer.

Modeling Excitability in Mechanosensory Neurons with MS Cation and MS Kv Channels

In mechanosensory neurons, depolarizing receptor potentials arise from mechanosensitive (MS) cation channels (ECation=∼0 mV), but new findings in touch and pain neurons indicate that firing characteristics are precisely modulated by MS Kv channels. Depending on the context in which it is elicited, mechanically facilitated Kv1 activation puts the brakes on mechanosensory firing thresholds or frequencies (Hao et al 2013 Neuron 77:899). This can, for instance, ensure that only intense (noxious) mechanical stimuli generate action potential (AP) traffic in high-threshold neurons. Here we use AP modeling to explore qualitatively how Kv channel mechanosensitivity could affect threshold behaviors and AP frequency characteristics. An important unsettled question is which transition(s) in the Kv channel's activation path accounts for Kv mechanosensitivity. Mechanosensitivity could reside with a voltage dependent step or with the thermal opening transition (Tabarean & Morris 2002 Biophys J 82:2982; Schmidt et al 2012 PNAS 109:10352). During membrane deformations this mechanistic “detail” would be consequential, we argue, because the MS transition's location can powerfully affect the magnitude of gKmax(apparent) and deactivation time course rates. To allow us computational access to the specific Kv transitions, we run a hybrid HH/Markov AP model. While the standard excitability machinery is given in HH terms, the MS conductances are described in Markov fashion. To mimic the effect of a membrane stretch deformation on the MS gK, the forward/backward rates of either the concerted voltage-dependent closed-closed transition or the thermal open-closed transition are increased/decreased reciprocally (e.g., doubled/halved). Mechanoreceptor neurons can exhibit phasic responsiveness to sustained mechanical stimuli; to explore such behavior, terms are included that allow for the possibility that the two MS conductances, MS gCation and the MS gK, experience deformation onset/offset intensities differently. Supported by NSERC Canada & by OHRI.

A Structure Based Course-Graining Physical Modeling of the Voltage Activated Kv1.2 Channel - Simulating and Analyzing the Fast Gating Current

K+ ion (Kv) channels catalyze ion permeation across membrane in a voltage dependent way, regulating electrical activities in cells. Several functional and structural studies demonstrate that Kv has a voltage sensor domain (VSD) that is coupled to changes in the membrane potential. An increase in the membrane potential activates the VSD, which drives the gate domain to open, generating voltage dependent ionic currents. There have been intense studies of the mechanism of the activation of Kv channels, using the kinetic models built from electrophysiological measurements. Recent structural studies also offered a detailed structural mechanism (with a phenomenological free energy landscape), suggesting how the movements of the conserved ARG residues in the VSD, upon changes in the membrane potential, can be facilitated. Starting with structural models of the voltage activation of the K1.2 channel and using our coarse-graining (CG) model allowed us to simulate the response of the channel to voltage changes. The simulations allowed us to evaluate the gating charge in terms of the change in the simulated electrolytes distribution. The change in gating charge was then converted to gating current using our renormalization model and the resulting time dependent of the protein fluctuation. The CG free energy landscapes allowed us to simulate the slow component of the gating current for the activation processes. Most significantly, our unique ability to simulate the time dependence of the fast gating current reproduces the observed trend, thereby providing a detailed picture of its molecular origin.

Expression and motor functional roles of voltage-dependent type 7 K+ channels in the human taenia coli

Voltage-dependent type 7 K+ (KV7 or KCNQ) channels modulate the excitability of neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators and the expression of KV7 channels in the human taenia coli. The effects of KV7 channel modulators on the muscle tone of human taenia coli strips were investigated under nonadrenergic non-nitrergic conditions by organ bath studies. Gene expression and tissue localisation of channels were studied by real-time PCR and immunohistochemistry, respectively. Under basal conditions, the KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean EC50 and Emax of 18.7μM and 30.5% respectively of the maximal bethanechol-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed the taenia coli, with mean EC50s of 19.2μM and 29.9μM, respectively. Retigabine also relaxed bethanechol-precontracted strips, with maximal relaxations of 79.2% of the bethanecol-induced precontraction. The motor effects induced by the KV7 channel modulators were not affected by tetrodotoxin or ω-conotoxin GVIA. XE-991 greatly reduced retigabine- and flupirtine-induced relaxations. Transcripts encoded by all KCNQ genes were detected in the taenia coli, with KCNQ4 showing the highest expression levels. KV7.4 channels were clearly visualised by immunohistochemistry in colonic epithelium, circular muscle layer and taenia coli. KV7 channels appear to contribute to the resting muscle tone of the human taenia coli. In addition, KV7 channel activators significantly relax the taenia coli. Thus, they could be useful therapeutic relaxant agents for colonic motor disorders.

Molecular Bases for the Asynchronous Activation of Sodium and Potassium Channels Required for Nerve Impulse Generation

Most action potentials are produced by the sequential activation of voltage-gated sodium (Nav) and potassium (Kv) channels. This is mainly achieved by the rapid conformational rearrangement of voltage-sensor (VS) modules in Nav channels, with activation kinetics up to 6-fold faster than Shaker-type Kv channels. Here, using mutagenesis and gating current measurements, we show that a 3-fold acceleration of the VS kinetics in Nav versus Shaker Kv channels is produced by the hydrophilicity of two "speed-control" residues located in the S2 and S4 segments in Nav domains I-III. An additional 2-fold acceleration of the Nav VS kinetics is provided by the coexpression of the β1 subunit, ubiquitously found in mammal tissues. This study uncovers the molecular bases responsible for the differential activation of Nav versus Kv channels, a fundamental prerequisite for the genesis of action potentials.

Control of Voltage-gated Potassium Channel Kv2.2 Expression by Pyruvate-Isocitrate Cycling Regulates Glucose-stimulated Insulin Secretion

Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet β-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of β-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 β-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.

Pathogenic plasticity of Kv7.2/3 channel activity is essential for the induction of tinnitus

Tinnitus, the perception of phantom sound, is often a debilitating condition that affects many millions of people. Little is known, however, about the molecules that participate in the induction of tinnitus. In brain slices containing the dorsal cochlear nucleus, we reveal a tinnitus-specific increase in the spontaneous firing rate of principal neurons (hyperactivity). This hyperactivity is observed only in noise-exposed mice that develop tinnitus and only in the dorsal cochlear nucleus regions that are sensitive to high frequency sounds. We show that a reduction in Kv7.2/3 channel activity is essential for tinnitus induction and for the tinnitus-specific hyperactivity. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. Our in vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. Together, our studies provide an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Moreover, our findings point to previously unknown biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans.

5-HT2 receptors-mediated modulation of voltage-gated K+ channels and neurophysiopathological correlates

The activity of voltage-gated K(+) channels (Kv) can be dynamically modulated by several events, including neurotransmitter stimulated biochemical cascades mediated by G protein-coupled receptors such as 5-HT2 receptors (5-HT2Rs). Activation of 5-HT2A/CR inhibits the Shaker-like K(+) channels Kv1.1 and Kv1.2, and this modulation involves the dual coordination of both RPTPα and distinct tyrosine kinases coupled to this receptor; 5-HT2Rs-mediated modulation of Kv channels controls glutamate release onto prefrontal cortex neurons that might play critical roles in neurophysiological, neurological, and psychiatric conditions. Noticeably, hallucinogens modulate Kv channel activity, acting at 5-HT2R. Hence, comprehensive knowledge of 5-HT2R signaling through modulation of distinct K(+) channels is a pivotal step in the direction that will enable scientists to discover novel 5-HT functions and dysfunctions in the brain and to identify original therapeutic targets.

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

Conserved N-Terminal Negative Charges Support Optimally Efficient N-type Inactivation of Kv1 Channels

N-type inactivation is produced by the binding of a potassium channel's N-terminus within the open pore, blocking conductance. Previous studies have found that introduction of negative charges into N-terminal inactivation domains disrupts inactivation; however, the Aplysia AKv1 N-type inactivation domain contains two negatively charged residues, E2 and E9. Rather than being unusual, sequence analysis shows that this N-terminal motif is highly conserved among Kv1 sequences across many phyla. Conservation analysis shows some tolerance at position 9 for other charged residues, like D9 and K9, whereas position 2 is highly conserved as E2. To examine the functional importance of these residues, site directed mutagenesis was performed and effects on inactivation were recorded by two electrode voltage clamp in Xenopus oocytes. We find that inclusion of charged residues at positions 2 and 9 prevents interactions with non-polar sites along the inactivation pathway increasing the efficiency of pore block. In addition, E2 appears to have additional specific electrostatic interactions that stabilize the inactivated state likely explaining its high level of conservation. One possible explanation for E2's unique importance, consistent with our data, is that E2 interacts electrostatically with a positive charge on the N-terminal amino group to stabilize the inactivation domain at the block site deep within the pore. Simple electrostatic modeling suggests that due to the non-polar environment in the pore in the blocked state, even a 1 Å larger separation between these charges, produced by the E2D substitution, would be sufficient to explain the 65× reduced affinity of the E2D N-terminus for the pore. Finally, our studies support a multi-step, multi-site N-type inactivation model where the N-terminus interacts deep within the pore in an extended like structure placing the most N-terminal residues 35% of the way across the electric field in the pore blocked state.

Stress peptide PACAP engages multiple signaling pathways within the carotid body to initiate excitatory responses in respiratory and sympathetic chemosensory afferents

Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca²⁺ mobilization, inhibition of various K⁺ channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (~41%), whereas the TASK-1-like K⁺ channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (~48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K⁺ channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation.