L-type Channel Cav1 Research Articles

Structure-Activity Relationship and Voltage Dependence for the Drug-Drug Interaction between Amiodarone Analogs and MNI-1 at the L-type Cav Channel.

The drug-drug interaction (DDI) between amiodarone (AMIO) and sofosbuvir (SOF), a direct-acting hepatitis-C NS5B nucleotide polymerase inhibitor, has been associated with severe bradyarrhythmia in patients. Recent cryo-EM data has revealed that this DDI occurs at the α-subunit of L-type Cav channels, with AMIO binding at the fenestration site and SOF [or MSD nucleotide inhibitor #1 (MNI-1): analog of SOF] binding at the central cavity of the conductance pathway. In this study, we investigated the DDI between 21 AMIO analogs, including dronedarone (DRON) and MNI-1 (or SOF) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hCav1.2 models. Our findings indicate that among the tested AMIO analogs in hiPSC-CMs at clinically relevant concentrations, only three analogs (AA-9, AA-10, and AA-17) were able to effectively substitute for AMIO in this DDI with 1 µM MNI-1. This highlights the importance of the diethyl amino group of AMIO for interacting with MNI-1. In the hCav1.2 model, desethylamiodarone (AA-12) demonstrated synergy with 90 µM MNI-1, while three other analogs with modifications to the position of the diethyl amino group or removal of iodo groups showed weaker synergy with 90 µM MNI-1. Interestingly, DRON did not exhibit any interaction with 270 µM SOF or 90 µM MNI-1, suggesting that it could safely replace AMIO in patients requiring SOF treatment, other clinically relevant differences considered. Overall, our functional data align with the cryo-EM data, highlighting that this DDI is dependent on the structure of AMIO and cardiomyocyte resting membrane potential. SIGNIFICANCE STATEMENT: Our findings point to specific residues in the AMIO molecule playing a critical role in the DDI between AMIO and MNI-1 (SOF analog), confirming cryo-EM results. Applied at clinically relevant AMIO's concentrations or projected MNI-1's concentrations at the resting potentials mimicking the sinoatrial node, this DDI significantly slowed down or completely inhibited the beating of hiPSC-CMs. Finally, these in vitro results support the safe replacement of AMIO (Cordarone) with DRON (Multaq) for patients requiring SOF treatment, other clinical caveats considered.

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

The T-type calcium channel CaV3.2 regulates insulin secretion in the pancreatic β-cell

Voltage-gated Ca2+ (CaV) channel dysfunction leads to impaired glucose-stimulated insulin secretion in pancreatic β-cells and contributes to the development of type-2 diabetes (T2D). The role of the low-voltage gated T-type CaV channels in β-cells remains obscure. Here we have measured the global expression of T-type CaV3.2 channels in human islets and found that gene expression of CACNA1H, encoding CaV3.2, is negatively correlated with HbA1c in human donors, and positively correlated with islet insulin gene expression as well as secretion capacity in isolated human islets. Silencing or pharmacological blockade of CaV3.2 attenuates glucose-stimulated cytosolic Ca2+ signaling, membrane potential, and insulin release. Moreover, the endoplasmic reticulum (ER) Ca2+ store depletion is also impaired in CaV3.2-silenced β-cells. The linkage between T-type (CaV3.2) and L-type CaV channels is further identified by the finding that the intracellular Ca2+ signaling conducted by CaV3.2 is highly dependent on the activation of L-type CaV channels. In addition, CACNA1H expression is significantly associated with the islet predominant L-type CACNA1C (CaV1.2) and CACNA1D (CaV1.3) genes in human pancreatic islets. In conclusion, our data suggest the essential functions of the T-type CaV3.2 subunit as a mediator of β-cell Ca2+ signaling and membrane potential needed for insulin secretion, and in connection with L-type CaV channels.

Distinct Gene Expression Patterns of Calcium Channels and Related Signaling Pathways Discovered in Lymphomas.

Cell surface calcium (Ca2+) channels permit Ca2+ ion influx, with Ca2+ taking part in cellular functions such as proliferation, survival, and activation. The expression of voltage-dependent Ca2+ (CaV) channels may modulate the growth of hematologic cancers. Profile analysis of Ca2+ channels, with a focus on the Ca2+ release-activated Ca2+ (CRAC) and L-type CaV channels, was performed on RNA sequencing data from lymphoma cell lines and samples derived from patients with diffuse large B cell lymphoma (DLBCL). CaV1.2 expression was found to be elevated in classical Hodgkin lymphoma (CHL) cell lines when compared to other B cell lymphoma cell lines. In contrast, CHL exhibited reduced expression of ORAI2 and STIM2. In our differential expression analysis comparing activated B cell-like DLBCL (ABC-DLBCL) and germinal centre B cell-like DLBCL (GCB-DLBCL) patient samples, ABC-DLBCL revealed stronger expression of CaV1.3, whereas CaV1.1, CaV1.2, and CaV1.4 showed greater expression levels in GCB-DLBCL. Interestingly, no differences in ORAI/STIM expression were noted in the patient samples. As Ca2+ is known to bind to calmodulin, leading to calcineurin activation and the passage of nuclear factor of activated T cells (NFAT) to the cell nucleus, pathways for calcineurin, calmodulin, NFAT, and Ca2+ signaling were also analyzed by gene set enrichment analysis. The NFAT and Ca2+ signaling pathways were found to be upregulated in the CHL cell lines relative to other B cell lymphoma cell lines. Furthermore, the calmodulin and Ca2+ signaling pathways were shown to be downregulated in the ABC-DLBCL patient samples. The findings of this study suggest that L-type CaV channels and Ca2+-related pathways could serve as differentiating components for biologic therapies in targeted lymphoma treatments.

Restoration of DNA integrity and the cell cycle by electric stimulation in planarian tissues damaged by ionizing radiation.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention.

Comment on “Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels”

Comment on “Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels”

S1928 Phosphorylation Tunes Vascular L‐type Channel Ca V 1.2 and Arterial Function during Angiotensin II Signaling and Hypertension

The L-type channel CaV1.2 is essential for vascular smooth muscle contraction and arterial tone. Increased vascular CaV1.2 expression and function are related to high smooth muscle contractility and enhanced arterial tone during hypertension, which is characterized by enhanced angiotensin II (angII) signaling. Several pathways have been proposed to account for increased CaV1.2 expression during hypertension, including increased CaV1.2 trafficking. However, mechanisms underlying enhanced CaV1.2 function during angII signaling and hypertension remain a subject of intense investigation. In this study, we hypothesize that CaV1.2 phosphorylation at the S1928 site is a key event that mediates increased channel activity and vascular reactivity during angII signaling and hypertension. Initial experiments found increased S1928 phosphorylation in angII-treated wild type arterial lysates. This was associated with elevated vascular smooth muscle whole-cell L-type channel currents, global intracellular Ca2+, and contraction. Similarly, ex vivo and in vivo experiments in mesenteric arteries reveal an increased arterial tone and decreased mesenteric blood flow from wild type mice. Moreover, smooth muscle cells treated with angII showed a redistribution of CaV1.2 into larger clusters. These functional changes were prevented or significantly ameliorated in arteries or cells from a knockin mouse expressing a mutant CaV1.2 in which serine was replaced with alanine at position 1928 (S1928A mouse) and by protein kinase C inhibition. In angII-induced hypertensive mice, increased CaV1.2 clusters size and channel activity, enhanced arterial tone, and mean arterial pressure in wild type mice were prevented or significantly reduce in S1928A mice. Altogether, these results suggest a key role for phosphorylation of CaV1.2 at S1928 in regulating CaV1.2 distribution, activity, and vascular function during angII signaling and hypertension. Phosphorylation of this single vascular CaV1.2 amino acid could be an essential regulatory mechanism that could be exploited for therapeutic intervention.

TSPAN-7 as a key regulator of glucose-stimulated Ca2+ influx and insulin secretion.

TSPAN-7 as a key regulator of glucose-stimulated Ca2+ influx and insulin secretion.

Ciguatera poisoning: the role of high-voltage-activated and store-operated calcium channels in ciguatoxin-induced sensory effects

Introduction: Ciguatera fish poisoning (CFP), the most common seafood poisoning worldwide, is caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Pruritus is one of the most distressing symptoms, associated with other cutaneous sensory disorders, including paresthesia and cold dysesthesia. No specific treatment exists. CTXs are known to primarily activate voltage-gated sodium channels, but the downstream molecular events that lead to sensory disturbances remain poorly defined. Peptidergic sensory neurons were recently identified as major players in CFP sensory disturbances. Methods: In this study, we examined the role of molecular actors in 2 effects induced by Pacific CTX-2 (P-CTX-2): the increase in cytosolic calcium levels in rat primary sensory neurons; and the release of the neuropeptide substance P (SP) in sensory neurons co-cultured with keratinocytes. Results: Our results (i) rule out the involvement of the Na+/Ca2+ exchanger (NCX) and the transient receptor potential channels transient receptor potential ankyrin 1 and and transient receptor potential vanilloid 1; (ii) show that N-type voltage-gated calcium (Cav) channels contribute to the initiation of the calcium signal elicited by P-CTX-2 in rat sensory neurons, while N-type and L-type Cav channels play equal parts in the SP release in the co-culture; and (iii) identify store-operated calcium entry supported by Orai calcium release-activated calcium modulator 1 (ORAI1) as a critical effector of the late phase of the calcium signal and the subsequent SP release elicited by P-CTX-2. Discussion: Our in vitro findings indicate that Cav and ORAI1 channels may be promising pharmacological targets for specifically relieving the sensory effects of CTXs.

Tetraspanin-7 regulation of L-type voltage-dependent calcium channels controls pancreatic β-cell insulin secretion.

Tetraspanin (TSPAN) proteins regulate many biological processes, including intracellular calcium (Ca2+ ) handling. TSPAN-7 is enriched in pancreatic islet cells; however, the function of islet TSPAN-7 has not been identified. Here, we characterize how β-cell TSPAN-7 regulates Ca2+ handling and hormone secretion. We find that TSPAN-7 reduces β-cell glucose-stimulated Ca2+ entry, slows Ca2+ oscillation frequency and decreases glucose-stimulated insulin secretion. TSPAN-7 controls β-cell function through a direct interaction with L-type voltage-dependent Ca2+ channels (CaV 1.2 and CaV 1.3), which reduces channel Ca2+ conductance. TSPAN-7 slows activation of CaV 1.2 and accelerates recovery from voltage-dependent inactivation; TSPAN-7 also slows CaV 1.3 inactivation kinetics. These findings strongly implicate TSPAN-7 as a key regulator in determining the set-point of glucose-stimulated Ca2+ influx and insulin secretion. Glucose-stimulated insulin secretion (GSIS) is regulated by calcium (Ca2+ ) entry into pancreatic β-cells through voltage-dependent Ca2+ (CaV ) channels. Tetraspanin (TSPAN) transmembrane proteins control Ca2+ handling, and thus they may also modulate GSIS. TSPAN-7 is the most abundant islet TSPAN and immunostaining of mouse and human pancreatic slices shows that TSPAN-7 is highly expressed in β- and α-cells; however, the function of islet TSPAN-7 has not been determined. Here, we show that TSPAN-7 knockdown (KD) increases glucose-stimulated Ca2+ influx into mouse and human β-cells. Additionally, mouse β-cell Ca2+ oscillation frequency was accelerated by TSPAN-7 KD. Because TSPAN-7 KD also enhanced Ca2+ entry when membrane potential was clamped with depolarization, the effect of TSPAN-7 on CaV channel activity was examined. TSPAN-7 KD enhanced L-type CaV currents in mouse and human β-cells. Conversely, heterologous expression of TSPAN-7 with CaV 1.2 and CaV 1.3 L-type CaV channels decreased CaV currents and reduced Ca2+ influx through both channels. This was presumably the result of a direct interaction of TSPAN-7 and L-type CaV channels because TSPAN-7 coimmunoprecipitated with both CaV 1.2 and CaV 1.3 from primary human β-cells and from a heterologous expression system. Finally, TSPAN-7 KD in human β-cells increased basal (5.6 mM glucose) and stimulated (45 mM KCl + 14 mM glucose) insulin secretion. These findings strongly suggest that TSPAN-7 modulation of β-cell L-type CaV channels is a key determinant of β-cell glucose-stimulated Ca2+ entry and thus the set-point of GSIS.

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

Cdk5 phosphorylates CaV1.3 channels and regulates GABAA-mediated miniature inhibitory post-synaptic currents in striato-nigral terminals

Neurotransmission is one of the most important processes in neuronal communication and depends largely on Ca2+ entering synaptic terminals through voltage-gated Ca2+ (CaV) channels. Although the contribution of L-type CaV channels in neurotransmission has not been unambiguously established, increasing evidence suggests a role for these proteins in noradrenaline, dopamine, and GABA release. Here we report the regulation of L-type channels by Cdk5, and its possible effect on GABA release in the substantia nigra pars reticulata (SNpr). Using patch-clamp electrophysiology, we show that Cdk5 inhibition by Olomoucine significantly increases current density through CaV1.3 (L-type) channels heterologously expressed in HEK293 cells. Likewise, in vitro phosphorylation showed that Cdk5 phosphorylates residue S1947 in the C-terminal region of the pore-forming subunit of CaV1.3 channels. Consistent with this, the mutation of serine into alanine (S1947A) prevented the regulation of Cdk5 on CaV1.3 channel activity. Our data also revealed that the inhibition of Cdk5 increased the frequency of high K+-evoked miniature inhibitory postsynaptic currents in rat SNpr neurons, acting on L-type channels. These results unveil a novel regulatory mechanism of GABA release in the SNpr that involves a direct action of Cdk5 on L-type channels.

Ca2+ -dependent and Ca2+ -independent somatic release from trigeminal neurons.

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca2+ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca2+ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca2+ influx, and a slower component triggered by Ca2+ influx. The Ca2+ -dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca2+ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.

Increased Calcium Influx through L-type Calcium Channels in Human and Mouse Neural Progenitors Lacking Fragile X Mental Retardation Protein.

SummaryThe absence of FMR1 protein (FMRP) causes fragile X syndrome (FXS) and disturbed FMRP function is implicated in several forms of human psychopathology. We show that intracellular calcium responses to depolarization are augmented in neural progenitors derived from human induced pluripotent stem cells and mouse brain with FXS. Increased calcium influx via nifedipine-sensitive voltage-gated calcium (Cav) channels contributes to the exaggerated responses to depolarization and type 1 metabotropic glutamate receptor activation. The ratio of L-type/T-type Cav channel expression is increased in FXS progenitors and correlates with enhanced progenitor differentiation to glutamate-responsive cells. Genetic reduction of brain-derived neurotrophic factor in FXS mouse progenitors diminishes the expression of Cav channels and activity-dependent responses, which are associated with increased phosphorylation of the phospholipase C-γ1 site within TrkB receptors and changes of differentiating progenitor subpopulations. Our results show developmental effects of increased calcium influx via L-type Cav channels in FXS neural progenitors.

Ion channel signaling influences cellular proliferation and phagocyte activity during axolotl tail regeneration

Little is known about the potential for ion channels to regulate cellular behaviors during tissue regeneration. Here, we utilized an amphibian tail regeneration assay coupled with a chemical genetic screen to identify ion channel antagonists that altered critical cellular processes during regeneration. Inhibition of multiple ion channels either partially (anoctamin1/Tmem16a, anoctamin2/Tmem16b, KV2.1, KV2.2, L-type CaV channels and H/K ATPases) or completely (GlyR, GABAAR, KV1.5 and SERCA pumps) inhibited tail regeneration. Partial inhibition of tail regeneration by blocking the calcium activated chloride channels, anoctamin1&2, was associated with a reduction of cellular proliferation in tail muscle and mesenchymal regions. Inhibition of anoctamin 1/2 also altered the post-amputation transcriptional response of p44/42 MAPK signaling pathway genes, including decreased expression of erk1/erk2. We also found that complete inhibition via voltage gated K+ channel blockade was associated with diminished phagocyte recruitment to the amputation site. The identification of H+ pumps as required for axolotl tail regeneration supports findings in Xenopus and Planaria models, and more generally, the conservation of ion channels as regulators of tissue regeneration. This study provides a preliminary framework for an in-depth investigation of the mechanistic role of ion channels and their potential involvement in regulating cellular proliferation and other processes essential to wound healing, appendage regeneration, and tissue repair.

AB317. SPR-44 Pharmacological activation of individual KCNQ channel subtypes in detrusor smooth muscle represents a promising novel approach for overactive bladder treatment

ObjectiveOur recent studies have demonstrated voltage-gated KCNQ channels (KCNQ1-KCNQ5) as key regulators of detrusor smooth muscle (DSM) function. Despite emerging developments, the physiological role of individual KCNQ channel subtypes remains less clear. Here, we utilized the novel compound ML-213, a potent activator of KCNQ2, KCNQ4, and KCNQ5 channels, to elucidate their physiological roles in guinea pig DSM function.MethodsUsing isometric DSM tension recordings, Ca2+ imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating DSM excitability and contractility.ResultsML-213 concentration-dependently (100 nM–30 µM) inhibited spontaneous phasic, pharmacologically-induced, and nerve-evoked contractions in DSM isolated strips. ML-213 (10 µM) decreased the global intracellular Ca2+ concentrations in DSM isolated strips, effects blocked by the L-type voltage-gated Ca2+ (CaV) channel inhibitor nifedipine (1 µM) and the KCNQ1-KCNQ5 channel inhibitor XE991 (10 µM). These data suggest that ML-213 decreases the global intracellular Ca2+ concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltage- and Ca2+-activated K+ channel inhibitor paxilline (1 µM) and gadolinium chloride (GdCl3, 50 µM), which blocks L-type CaV channels and non-selective cation channels. Under these experimental conditions, ML-213 (10 µM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K+ transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca2+ concentration and DSM contractility.ConclusionsThese data using the novel compound ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability, intracellular Ca2+ concentration, and contractility of DSM by virtue of their control of the membrane potential. Moreover, these new findings provide a foundational basis for future investigations on KCNQ channel functional roles in human DSM excitability and contractility to confirm their potential as novel therapeutic targets for overactive bladder.Source of FundingNIH grant R01-DK106964 to GV Petkov and F31-DK104528 to A Provence.

MP30-06 IDENTIFICATION OF A DIVERSE FUNGAL COMMUNITY (“MYCOBIOME”) IN THE NORMAL FEMALE HUMAN LOWER URINARY TRACT

METHODS: Using isometric DSM tension recordings, Ca imaging, and amphotericin-B perforated patch-clamp electrophysiology, we elucidated the role of ML-213-sensitive KCNQ channels in regulating guinea pig DSM excitability and contractility. RESULTS: ML-213 concentration-dependently (100 nM-30 mM) inhibited spontaneous phasic, pharmacologically-induced, and nerveevoked contractions in DSM isolated strips. ML-213 (10 mM) decreased the global intracellular Ca concentration and inhibited spontaneous Ca transients, effects blocked by the L-type voltage-gated Ca (CaV) channel inhibitor nifedipine (1 mM) and the KCNQ1KCNQ5 channel inhibitor XE991 (10 mM). These data suggests that ML-213 decreases the global intracellular Ca concentration by inhibiting L-type CaV channels through an indirect mechanism downstream from KCNQ channel activation. In addition, ML-213 hyperpolarized the cell membrane potential and inhibited spontaneous action potentials in DSM cells, effects reversible by washout. We next aimed to examine the effects of ML-213 on whole cell KCNQ currents. To isolate KCNQ currents, the bath solution contained the large conductance voltageand Ca-activated K channel inhibitor paxilline (1 mM) and gadolinium chloride (GdCl3, 100 mM), which blocks L-type CaV channels and nonselective cation channels. Under these experimental conditions, ML213 (10 mM) enhanced whole cell KCNQ currents. These findings suggest that the modulation of K transport through ML-213-sensitive KCNQ channels underlies ML-213-induced cell membrane hyperpolarization to decrease the global intracellular Ca concentration and DSM contractility. CONCLUSIONS: These results, using the novel KCNQ channel opener ML-213, suggest that KCNQ2-, KCNQ4-, and KCNQ5-containing channels are essential regulators of the excitability and contractility of DSM, and therefore could represent novel molecular targets for pharmacological or genetic control of overactive bladder.

Dendritic sodium spikes are required for long-term potentiation at distal synapses on hippocampal pyramidal neurons.

Dendritic integration of synaptic inputs mediates rapid neural computation as well as longer-lasting plasticity. Several channel types can mediate dendritically initiated spikes (dSpikes), which may impact information processing and storage across multiple timescales; however, the roles of different channels in the rapid vs long-term effects of dSpikes are unknown. We show here that dSpikes mediated by Nav channels (blocked by a low concentration of TTX) are required for long-term potentiation (LTP) in the distal apical dendrites of hippocampal pyramidal neurons. Furthermore, imaging, simulations, and buffering experiments all support a model whereby fast Nav channel-mediated dSpikes (Na-dSpikes) contribute to LTP induction by promoting large, transient, localized increases in intracellular calcium concentration near the calcium-conducting pores of NMDAR and L-type Cav channels. Thus, in addition to contributing to rapid neural processing, Na-dSpikes are likely to contribute to memory formation via their role in long-lasting synaptic plasticity.

Altered voltage-gated calcium channels in rat inferior colliculus neurons contribute to alcohol withdrawal seizures

We have previously reported that enhanced susceptibility to alcohol withdrawal seizures (AWS) parallels the enhancement of the current density of high-threshold voltage-gated Ca2+ (CaV) channels in rat inferior colliculus (IC) neurons. However, whether this increased current density is a cause or consequence of AWS is unclear. Here, I report changes in the current density of CaV channels in IC neurons during the course of alcohol withdrawal and the potential anticonvulsant effect of intra-IC infusions of L- and P-type CaV channel antagonists. Whole-cell currents were activated by depolarizing pulses using barium as the charge carrier. Currents and seizure susceptibility were evaluated in control animals 3h after alcohol intoxication, as well as 3h (before AWS), 24h (when AWS susceptibility is maximal), and 48h (when AWS susceptibility is no longer present) after alcohol withdrawal. Nifedipine, nimodipine (L-type antagonists) or ω-agatoxin TK (P-type antagonist) were infused intra-IC to probe the role of CaV channels in the pathogenesis of AWS. CaV current density and conductance in IC neurons were significantly increased 3 and 24h after alcohol withdrawal compared with the control group or the group tested 3h following ethanol intoxication. Blockade of L-type CaV channels within the IC completely suppressed AWS, and inhibition of P-type channels reduced AWS severity. These findings suggest that the enhancement of CaV currents in IC neurons occurs prior to AWS onset and that alterations in L- and P-type CaV channels in these neurons may underlie the pathogenesis of AWS.

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca2+ into cells by CaV channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca2+ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (CaV2.2) channels for permeating Ca2+ and Ba2+ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when CaV currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (CaV1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba2+ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through CaV1 and CaV2 channels (i.e. high-voltage activated CaV channels). The voltage dependence of CaV1 channel permeation is likely a mechanism mediating sustained Ca2+ influx during the plateau phase of the cardiac action potential.