Vestibular Role of KCNQ4 and KCNQ5 K+ Channels Revealed by Mouse Models

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K⁺ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (K_v7.4) K⁺ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K⁺ efflux. Like the related K⁺ channel KCNQ5 (K_v7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4^−/− mice lacking KCNQ4, as well as Kcnq4^dn/dn and Kcnq5^dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4^−/−, Kcnq5^dn/dn nor Kcnq4^−/−/Kcnq5^dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4^−/−/Kcnq5^dn/dn and Kcnq4^−/− mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K⁺ removal and modulation of synaptic transmission.     

The Identification of Histidine 712 as a Critical Residue for Constitutive TRPV5 Internalization

The epithelial Ca²⁺ channel TRPV5 constitutes the apical entry gate for Ca²⁺ transport in renal epithelial cells. Ablation of the trpv5 gene in mice leads to a reduced Ca²⁺ reabsorption. TRPV5 is tightly regulated by various calciotropic hormones, associated proteins, and other factors, which mainly affect channel activity via the C terminus. To further identify the role of the C terminus in TRPV5 regulation, we expressed channels harboring C-terminal deletions and studied channel activity by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using fura-2 analysis. Removal of amino acid His⁷¹² elevated the [Ca²⁺]_i, indicating enlarged TRPV5 activity. In addition, substitution of the positively charged His⁷¹² for a negative (H712D) or neutral (H712N) amino acid also stimulated TRPV5 activity. This critical role of His⁷¹² was confirmed by patch clamp analysis, which demonstrates increased Na⁺ and Ca²⁺ currents for TRPV5-H712D. Cell surface biotinylation studies revealed enhanced plasma membrane expression of TRPV5-H712D as compared with wild-type (WT) TRPV5. This elevated plasma membrane presence also was observed with the Ca²⁺-impermeable TRPV5-H712D and TRPV5-WT pore mutants, demonstrating that the elevation is not due to the increased [Ca²⁺]_i. Finally, using an internalization assay, we demonstrated a delayed cell surface retrieval for TRPV5-H712D, likely causing the increase in plasma membrane expression. Together, these results demonstrate that His⁷¹² plays an essential role in plasma membrane regulation of TRPV5 via a constitutive endocytotic mechanism.     

Calretinin Regulates Ca2+-dependent Inactivation and Facilitation of Cav2.1 Ca2+ Channels through a Direct Interaction with the α12.1 Subunit

Voltage-gated Ca_v2.1 Ca²⁺ channels undergo dual modulation by Ca²⁺, Ca²⁺-dependent inactivation (CDI), and Ca²⁺-dependent facilitation (CDF), which can influence synaptic plasticity in the nervous system. Although the molecular determinants controlling CDI and CDF have been the focus of intense research, little is known about the factors regulating these processes in neurons. Here, we show that calretinin (CR), a Ca²⁺-binding protein highly expressed in subpopulations of neurons in the brain, inhibits CDI and enhances CDF by binding directly to α₁2.1. Screening of a phage display library with CR as bait revealed a highly basic CR-binding domain (CRB) present in multiple copies in the cytoplasmic linker between domains II and III of α₁2.1. In pulldown assays, CR binding to fusion proteins containing these CRBs was largely Ca²⁺-dependent. α₁2.1 coimmunoprecipitated with CR antibodies from transfected cells and mouse cerebellum, which confirmed the existence of CR-Ca_v2.1 complexes in vitro and in vivo. In HEK293T cells, CR significantly decreased Ca_v2.1 CDI and increased CDF. CR binding to α₁2.1 was required for these effects, because they were not observed upon substitution of the II-III linker of α₁2.1 with that from the Ca_v1.2 α₁ subunit (α₁1.2), which lacks the CRBs. In addition, coexpression of a protein containing the CRBs blocked the modulatory action of CR, most likely by competing with CR for interactions with α₁2.1. Our findings highlight an unexpected role for CR in directly modulating effectors such as Ca_v2.1, which may have major consequences for Ca²⁺ signaling and neuronal excitability.     

Functional Significance of K+ Channel β-Subunit KCNE3 in Auditory Neurons

The KCNE3 β-subunit interacts with and regulates the voltage-dependent gating, kinetics, and pharmacology of a variety of Kv channels in neurons. Because a single neuron may express multiple KCNE3 partners, it is impossible to predict the overall functional relevance of the single transmembrane domain peptide on the pore-forming K⁺ channel subunits with which it associates. In the inner ear, the role of KCNE3 is undefined, despite its association with Meniere disease and tinnitus. To gain insights on the functional significance of KCNE3 in auditory neurons, we examined the properties of spiral ganglion neurons (SGNs) in Kcne3 null mutant neurons relative to their age-matched controls. We demonstrate that null deletion of Kcne3 abolishes characteristic wide variations in the resting membrane potentials of SGNs and yields age-dependent alterations in action potential and firing properties of neurons along the contour of the cochlear axis, in comparison with age-matched wild-type neurons. The properties of basal SGNs were markedly altered in Kcne3^−/− mice compared with the wild-type controls; these include reduced action potential latency, amplitude, and increased firing frequency. Analyses of the underlying conductance demonstrate that null mutation of Kcne3 results in enhanced outward K⁺ currents, which is sufficient to explain the ensuing membrane potential changes. Additionally, we have demonstrated that KCNE3 may regulate the activity of Kv4.2 channels in SGNs. Finally, there were developmentally mediated compensatory changes that occurred such that, by 8 weeks after birth, the electrical properties of the null mutant neurons were virtually indistinguishable from the wild-type neurons, suggesting that ion channel remodeling in auditory neurons progresses beyond hearing onset.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

A dynamic model of saliva secretion

We construct a mathematical model of the parotid acinar cell with the aim of investigating how the distribution of K⁺ and Cl⁻ channels affects saliva production. Secretion of fluid is initiated by Ca²⁺ signals acting on Ca²⁺ dependent K⁺ and Cl⁻ channels. The opening of these channels facilitates the movement of Cl⁻ ions into the lumen which water follows by osmosis. We use recent results into both the release of Ca²⁺ from internal stores via the inositol (1,4,5)-trisphosphate receptor (IP₃R) and IP₃ dynamics to create a physiologically realistic Ca²⁺ model which is able to recreate important experimentally observed behaviours seen in parotid acinar cells. We formulate an equivalent electrical circuit diagram for the movement of ions responsible for water flow which enables us to calculate and include distinct apical and basal membrane potentials to the model. We show that maximum saliva production occurs when a small amount of K⁺ conductance is located at the apical membrane, with the majority in the basal membrane. The maximum fluid output is found to coincide with a minimum in the apical membrane potential. The traditional model whereby all Cl⁻ channels are located in the apical membrane is shown to be the most efficient Cl⁻ channel distribution.     

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Low voltage-activated T-type Ca_v3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca_v3.2-dependent conductance. However, Ca_v3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca_v3.2 exists, there are no reports testing the specific role of Ca_v3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca_v3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca²⁺-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca_v3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.     

Aquaporin-1 Tunes Pain Perception by Interaction with Nav1.8 Na+ Channels in Dorsal Root Ganglion Neurons

Aquaporin-1 (AQP1) water channels are expressed in the plasma membrane of dorsal root ganglion (DRG) neurons. We found reduced osmotic water permeability in freshly isolated DRG neurons from AQP1^−/− versus AQP1^+/+ mice. Behavioral studies showed greatly reduced thermal inflammatory pain perception in AQP1^−/− mice evoked by bradykinin, prostaglandin E₂, and capsaicin as well as reduced cold pain perception. Patch clamp of freshly isolated DRG neurons showed reduced action potential firing in response to current injections. Single action potentials after pulse current injections showed reduced maximum inward current, suggesting impaired Na_v1.8 Na⁺ function. Whole-cell Na_v1.8 Na⁺ currents in Na_v1.8-expressing ND7-23 cells showed slowed frequency-dependent inactivation after AQP1 transfection. Immunoprecipitation studies showed AQP1- Na_v1.8 Na⁺ interaction, which was verified in live cells by single-particle tracking of quantum dot-labeled AQP1. Our results implicate the involvement of AQP1 in DRG neurons for the perception of inflammatory thermal pain and cold pain, whose molecular basis is accounted for, in part, by reduced Na_v1.8-dependent membrane Na⁺ current. AQP1 is, thus, a novel target for pain management.     

Reactive Oxygen Species-mediated TRPC6 Protein Activation in Vascular Myocytes,a Mechanism for Vasoconstrictor-regulated Vascular Tone

Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca²⁺ entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca²⁺ entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca²⁺ entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H₂O₂ itself evoked Ca²⁺ influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H₂O₂ also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H₂O₂ stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H₂O₂ triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.     

ACTH Induces Cav3.2 Current and mRNA by cAMP-dependent and cAMP-independent Mechanisms

Bovine adrenal zona fasciculata (AZF) cells express Ca_v3.2 T-type Ca²⁺ channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca_v3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3–6 days markedly enhanced the expression of Ca_v3.2 current. The increase in Ca_v3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Ca_v3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 μm) and 6-benzoyladenosine cAMP (300 μm) induced CACNA1H mRNA, but not Ca_v3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10–50 μm) enhanced CACNA1H mRNA and Ca_v3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Ca_v3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Ca_v3.2 current and mRNA with a potency and effectiveness similar to the parent compound. The Epac activator 8CPT-2′-O-methyl-cAMP and its metabolites 8CPT-2′-OMe-5′-AMP and 8CPT-2′-O-methyl-adenosine increased CACNA1H mRNA and Ca_v3.2 current; Sp-8CPT-2′-O-methyl-cAMP increased neither Ca_v3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca²⁺ current. Specifically, ACTH induces expression of CACNA1H mRNA and Ca_v3.2 current in AZF cells by mechanisms that depend at most only partly on cAMP. In contrast, cAMP enhances expression of CACNA1H mRNA but not the corresponding Ca²⁺ current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Ca_v3.2 current indirectly through metabolites. ACTH and the metabolites may induce Ca_v3.2 expression by the same, unidentified mechanism.     

Loss of the K+ channel Kv2.1 greatly reduces outward dark current and causes ionic dysregulation and degeneration in rod photoreceptors

Vertebrate retinal photoreceptors signal light by suppressing a circulating “dark current” that maintains their relative depolarization in the dark. This dark current is composed of an inward current through CNG channels and NCKX transporters in the outer segment that is balanced by outward current exiting principally from the inner segment. It has been hypothesized that K_v2.1 channels carry a predominant fraction of the outward current in rods. We examined this hypothesis by comparing whole cell, suction electrode, and electroretinographic recordings from K_v2.1 knockout (K_v2.1^−/−) and wild-type (WT) mouse rods. Single cell recordings revealed flash responses with unusual kinetics, and reduced dark currents that were quantitatively consistent with the measured depolarization of the membrane resting potential in the dark. A two-compartment (outer and inner segment) physiological model based on known ionic mechanisms revealed that the abnormal K_v2.1^−/− rod photoresponses arise principally from the voltage dependencies of the known conductances and the NCKX exchanger, and a highly elevated fraction of inward current carried by Ca²⁺ through CNG channels due to the aberrant depolarization. K_v2.1^−/− rods had shorter outer segments than WT and dysmorphic mitochondria in their inner segments. Optical coherence tomography of knockout animals demonstrated a slow photoreceptor degeneration over a period of 6 mo. Overall, these findings reveal that K_v2.1 channels carry 70–80% of the non-NKX outward dark current of the mouse rod, and that the depolarization caused by the loss of K_v2.1 results in elevated Ca²⁺ influx through CNG channels and elevated free intracellular Ca²⁺, leading to progressive degeneration.     

Network dynamics in nociceptive pathways assessed by the neuronal avalanche model

Background

Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.

Findings

When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K⁺ channels, K_v1.1 and K_v1.2 (voltage-gated K⁺ channels) and TREK1 and TRAAK (K_2P channels). IQ effectively reduced the currents mediated by both K⁺ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K_v1.1 and K_v1.2.

Conclusions

Our results demonstrate that IQ blocks the voltage-gated K⁺ channels to increase AP duration and K_2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.     

7,8,3'-Trihydroxyflavone, a potent small molecule TrkB receptor agonist, protects spiral ganglion neurons from degeneration both in vitro and in vivo

Most sensorineural hearing loss cases occur as a result of hair cell loss, which results in secondary degeneration of spiral ganglion neurons (SGNs). Substantial loss of SGNs reduces the benefit of cochlear implants, which rely on SGNs for transmitting signals to the central auditory centers. Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) play essential roles in cochlear development and are required for SGN survival. Here we report that 7,8,3'-trihydroxyflavone (7,8,3'-THF), which is a small molecule agonist of tyrosine receptor kinase B (TrkB), promoted SGN survival with high potency both in vitro and in vivo. The compound protected the SGNs in a TrkB-dependent manner, as its effects on SGNs disappeared when the TrkB was blocked. Application of 7,8,3'-THF in the bulla of conditional connexin26 (cCx26)-null mice dramatically rescued SGNs in the applied ear compared to untreated control cochlea in the same animal. Our findings suggest that 7,8,3'-THF is a promising therapeutic agent protecting the SGNs from degeneration both in vitro and in vivo.     

Ca<Subscript>v</Subscript>1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca²⁺ channels (LTCCs, Ca_v1) open readily during membrane depolarization and allow Ca²⁺ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca²⁺-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Ca_v1.2 and Ca_v1.3), Ca_v1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Ca_v1.3^−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Ca_v1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Ca_v1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Ca_v1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.     

Chronic fluoxetine administration increases expression of the L-channel gene Cav1.2 in astrocytes from the brain of treated mice and in culture and augments K-induced increase in [Ca]i

We have recently shown that freshly isolated astrocytes from the mouse brain express mRNA for the L-channel gene Ca_v1.3 to at least the same degree (per mg mRNA) as corresponding neurons. The amount of extracellular Ca²⁺ actually entering cultured astrocytes by its opening is modest, but due to secondary Ca²⁺-mediated stimulation of the ryanodine receptor (RyR) the increase in free cytosolic Ca²⁺ [Ca²⁺]_i is substantial. The other Ca_v1 subtype expressed in brain is Ca_v1.2, which is even expressed in higher density. Although the different primers used for the two genes preclude exact quantitative comparison, the present study suggests that this is also the case in the freshly isolated astrocytes and neurons, which express equal Ca_v1.2 densities. Again, most of the increase in [Ca²⁺]_i occurred by RyR activity. In contrast to Ca_v1.3 the expression of Ca_v1.2 was greatly increased (doubled) after two weeks of treatment with fluoxetine hydrochloride (10 mg/kg). Accordingly [Ca²⁺]_i in cultured astrocytes exposed to the addition of 10–60 mM KCl increased substantially in cultured astrocytes treated chronically with fluoxetine with the lag time until the effect was observed depending upon the fluoxetine concentration. This effect was inhibited by nifedipine or siRNA against Ca_v1.2. The increase in K⁺-induced rise in [Ca²⁺]_i after fluoxetine treatment is directly opposite to a decrease in [Ca²⁺]_i after treatment with any of the anti-bipolar drugs lithium, carbamazepine or valproic acid, due to reduced capacitative Ca²⁺ influx. We have previously shown a similar effect after fluoxetine treatment, but it becomes overridden by the Ca_v1.2 up-regulation.