RESUMEN
Skeletal muscle contraction triggers the exercise pressor reflex (EPR) to regulate the cardiovascular system response to exercise. During muscle contraction, substances are released that generate action potential activity in group III and IV afferents that mediate the EPR. Some of these substances increase afferent activity via G-protein-coupled receptor (GPCR) activation, but the mechanisms are incompletely understood. We were interested in determining if tetrodotoxin-resistant (TTX-R) voltage-dependent sodium channels (NaV) were involved and investigated the effect of a mixture of such compounds (bradykinin, prostaglandin, norepinephrine, and ATP, called muscle metabolites). Using whole cell patch-clamp electrophysiology, we show that the muscle metabolites significantly increased TTX-R NaV currents. The rise time of this enhancement averaged â¼2 min, which suggests the involvement of a diffusible second messenger pathway. The effect of muscle metabolites on the current-voltage relationship, channel activation and inactivation kinetics support NaV1.9 channels as the target for this enhancement. When applied individually at the concentration used in the mixture, only prostaglandin and bradykinin significantly enhanced NaV current, but the sum of these enhancements was <1/3 that observed when the muscle metabolites were applied together. This suggests synergism between the activated GPCRs to enhance NaV1.9 current. When applied at a higher concentration, all four substances could enhance the current, which demonstrates that the GPCRs activated by each metabolite can enhance channel activity. The enhancement of NaV1.9 channel activity is a likely mechanism by which GPCR activation increases action potential activity in afferents generating the EPR.NEW & NOTEWORTHY G-protein-coupled receptor (GPCR) activation increases action potential activity in muscle afferents to produce the exercise pressor reflex (EPR), but the mechanisms are incompletely understood. We provide evidence that NaV1.9 current is synergistically enhanced by application of a mixture of metabolites potentially released during muscle contraction. The enhancement of NaV1.9 current is likely one mechanism by which GPCR activation generates the EPR and the inappropriate activation of the EPR in patients with cardiovascular disease.
Asunto(s)
Bradiquinina , Ganglios Espinales , Canal de Sodio Activado por Voltaje NAV1.9/metabolismo , Adenosina Trifosfato/metabolismo , Bradiquinina/farmacología , Ganglios Espinales/fisiología , Humanos , Músculos , Neuronas Aferentes/fisiología , Norepinefrina/farmacología , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
The exercise pressor reflex (EPR) originates in skeletal muscle and is activated by exercise-induced signals to increase arterial blood pressure and cardiac output. Muscle ischemia can elicit the EPR, which can be inappropriately activated in patients with peripheral vascular disease or heart failure to increase the incidence of myocardial infarction. We seek to better understand the receptor/channels that control excitability of group III and group IV muscle afferent fibers that give rise to the EPR. Bradykinin (BK) is released within contracting muscle and can evoke the EPR. However, the mechanism is incompletely understood. KV7 channels strongly regulate neuronal excitability and are inhibited by BK. We have identified KV7 currents in muscle afferent neurons by their characteristic activation/deactivation kinetics, enhancement by the KV7 activator retigabine, and block by KV7 specific inhibitor XE991. The blocking of KV7 current by different XE991 concentrations suggests that the KV7 current is generated by both KV7.2/7.3 (high affinity) and KV7.5 (low affinity) channels. The KV7 current was inhibited by 300 nM BK in neurons with diameters consistent with both group III and group IV afferents. The inhibition of KV7 by BK could be a mechanism by which this metabolic mediator generates the EPR. Furthermore, our results suggest that KV7 channel activators such as retigabine, could be used to reduce cardiac stress resulting from the exacerbated EPR in patients with cardiovascular disease.NEW & NOTEWORTHY KV7 channels control neuronal excitability. We show that these channels are expressed in muscle afferents and generate currents that are blocked by XE991 and bradykinin (BK). The XE991 block suggests that KV7 current is generated by KV7.2/3 and KV7.5 channels. The BK inhibition of KV7 channels may explain how BK activates the exercise pressor reflex (EPR). Retigabine can enhance KV7 current, which could help control the inappropriately activated EPR in patients with cardiovascular disease.
Asunto(s)
Canales de Potasio KCNQ/fisiología , Contracción Muscular/fisiología , Músculo Esquelético/fisiología , Condicionamiento Físico Animal/fisiología , Reflejo/fisiología , Animales , Antracenos/farmacología , Anticonvulsivantes/farmacología , Carbamatos/farmacología , Relación Dosis-Respuesta a Droga , Canales de Potasio KCNQ/antagonistas & inhibidores , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Fenilendiaminas/farmacología , Ratas , Ratas Sprague-Dawley , Reflejo/efectos de los fármacosRESUMEN
The exercise pressor reflex (EPR) is activated by muscle contractions to increase heart rate and blood pressure during exercise. While this reflex is beneficial in healthy individuals, the reflex activity is exaggerated in patients with cardiovascular disease, which is associated with increased mortality. Group III and IV afferents mediate the EPR and have been shown to express both tetrodotoxin-sensitive (TTX-S, NaV1.6, and NaV1.7) and -resistant (TTX-R, NaV1.8, and NaV1.9) voltage-gated sodium (NaV) channels, but NaV1.9 current has not yet been demonstrated. Using a F--containing internal solution, we found a NaV current in muscle afferent neurons that activates at around -70 mV with slow activation and inactivation kinetics, as expected from NaV1.9 current. However, this current ran down with time, which resulted, at least in part, from increased steady-state inactivation since it was slowed by both holding potential hyperpolarization and a depolarized shift of the gating properties. We further show that, following NaV1.9 current rundown (internal F-), application of the NaV1.8 channel blocker A803467 inhibited significantly more TTX-R current than we had previously observed (internal Cl-), which suggests that NaV1.9 current did not rundown with that internal solution. Using immunohistochemistry, we found that the majority of group IV somata and axons were NaV1.9 positive. The majority of small diameter myelinated afferent somata (putative group III) were also NaV1.9 positive, but myelinated muscle afferent axons were rarely labeled. The presence of NaV1.9 channels in muscle afferents supports a role for these channels in activation and maintenance of the EPR. NEW & NOTEWORTHY Small diameter muscle afferents signal pain and muscle activity levels. The muscle activity signals drive the cardiovascular system to increase muscle blood flow, but these signals can become exaggerated in cardiovascular disease to exacerbate cardiac damage. The voltage-dependent sodium channel NaV1.9 plays a unique role in controlling afferent excitability. We show that NaV1.9 channels are expressed in muscle afferents, which supports these channels as a target for drug development to control hyperactivity of these neurons.
Asunto(s)
Axones/fisiología , Canal de Sodio Activado por Voltaje NAV1.9/metabolismo , Neuronas Aferentes/fisiología , Reflejo de Estiramiento/fisiología , Potenciales de Acción/fisiología , Compuestos de Anilina/farmacología , Animales , Furanos/farmacología , Ganglios Espinales/diagnóstico por imagen , Ganglios Espinales/fisiología , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Contracción Muscular/fisiología , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/fisiología , Distribución Normal , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.
Asunto(s)
Dolor en Cáncer/tratamiento farmacológico , Hiperalgesia/tratamiento farmacológico , Péptidos/administración & dosificación , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Analgésicos Opioides/efectos adversos , Animales , Dolor en Cáncer/inducido químicamente , Dolor en Cáncer/genética , Dolor en Cáncer/patología , Humanos , Hiperalgesia/inducido químicamente , Hiperalgesia/genética , Hiperalgesia/patología , Ligandos , Ratones , Ratones Noqueados , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Neuralgia/genética , Neuralgia/patología , Antagonistas Nicotínicos/administración & dosificación , Compuestos Organoplatinos/efectos adversos , Oxaliplatino , Receptores de GABA-B/genéticaRESUMEN
Patients with intermittent claudication suffer from both muscle pain and an exacerbated exercise pressor reflex. Excitability of the group III and group IV afferent fibers mediating these functions is controlled in part by voltage-dependent sodium (NaV) channels. We previously found tetrodotoxin-resistant NaV1.8 channels to be the primary type in muscle afferent somata. However, action potentials in group III and IV afferent axons are blocked by TTX, supporting a minimal role of NaV1.8 channels. To address these apparent differences in NaV channel expression between axon and soma, we used immunohistochemistry to identify the NaV channels expressed in group IV axons within the gastrocnemius muscle and the dorsal root ganglia sections. Positive labeling by an antibody against the neurofilament protein peripherin was used to identify group IV neurons and axons. We show that >67% of group IV fibers express NaV1.8, NaV1.6, or NaV1.7. Interestingly, expression of NaV1.8 channels in group IV somata was significantly higher than in the fibers, whereas there were no significant differences for either NaV1.6 or NaV1.7. When combined with previous work, our results suggest that NaV1.8 channels are expressed in most group IV axons, but that, under normal conditions, NaV1.6 and/or NaV1.7 play a more important role in action potential generation to signal muscle pain and the exercise pressor reflex.
Asunto(s)
Neuronas Aferentes/metabolismo , Células Receptoras Sensoriales/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Anticuerpos/metabolismo , Axones/efectos de los fármacos , Axones/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Masculino , Músculos/efectos de los fármacos , Músculos/metabolismo , Neuronas Aferentes/efectos de los fármacos , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Ganglio Cervical Superior/efectos de los fármacos , Ganglio Cervical Superior/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.
Asunto(s)
Conotoxinas/química , Caracol Conus/química , Antagonistas Nicotínicos/química , Receptores Nicotínicos/química , Amidas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Canales de Calcio/química , Clonación Molecular , Retículo Endoplásmico/metabolismo , Hiperalgesia/tratamiento farmacológico , Concentración 50 Inhibidora , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Neuralgia/terapia , Oocitos/citología , Conformación Proteica , Señales de Clasificación de Proteína , Ratas , Ratas Sprague-Dawley , Xenopus laevisRESUMEN
Chronic pain is very difficult to treat. Thus, novel analgesics are a critical area of research. Strong pre-clinical evidence supports the analgesic effects of α-conopeptides, Vc1.1 and RgIA, which block α9α10 nicotinic acetylcholine receptors (nAChRs). However, the analgesic mechanism is controversial. Some evidence supports the block of α9α10 nAChRs as an analgesic mechanism, while other evidence supports the inhibition of N-type CaV (CaV2.2) current via activation of GABAB receptors. Here we reassess the effect of Vc1.1 and RgIA on CaV current in rat sensory neurons. Unlike the previous findings, we found highly variable effects among individual sensory neurons, but on average only minimal inhibition induced by Vc1.1, and no significant effect on the current by RgIA. We also investigated the potential involvement of GABAB receptors in the Vc1.1 induced inhibition, and found no correlation between the size of CaV current inhibition induced by baclofen (GABAB agonist) vs. that induced by Vc1.1. Thus, GABAB receptors are unlikely to mediate the Vc1.1 induced CaV current inhibition. Based on the present findings, CaV current inhibition in dorsal root ganglia is unlikely to be the predominant mechanism by which either Vc1.1 or RgIA induce analgesia. SIGNIFICANCE STATEMENT: Better analgesic drugs are desperately needed to help physicians to treat pain. While many pre-clinical studies support the analgesic effects of α-conopeptides, Vc1.1 and RgIA, the mechanism is controversial. The development of improved α-conopeptide analgesics would be greatly facilitated by a complete understanding of the analgesic mechanism. However, we show that we cannot reproduce one of the proposed analgesic mechanisms, which is an irreversible inhibition of CaV current in a majority of sensory neurons.
RESUMEN
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.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/metabolismo , Permeabilidad de la Membrana Celular , ElectrofisiologíaRESUMEN
Voltage-gated calcium (Ca(V)) channels deliver Ca(2+) to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca(2+) over other cations is thought to involve multiple Ca(2+)-binding sites within the pore. Although the Ca(2+) affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (Ca(V)2.2) channels to investigate the effect of voltage on Ca(2+) flux. We found that the EC50 for Ca(2+) permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca(2+) ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow Ca(V)2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca(2+)-Ba(2+) anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca(2+)-Ba(2+) anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca(2+) permeation through Ca(V)2.2 channels may require at least four Ca(2+)-binding sites. Finally, our results suggest that the high affinity of Ca(2+) for the channel helps to enhance Ca(2+) influx at depolarized voltages relative to other ions (e.g., Ba(2+) or Na(+)), whereas the absence of voltage effects at negative potentials prevents Ca(2+) from becoming a channel blocker. Both effects are needed to maximize Ca(2+) influx over the voltages spanned by action potentials.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Potenciales de la Membrana , Animales , Bario/farmacología , Sitios de Unión , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/química , Células Cultivadas , Transporte Iónico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Purinas/farmacología , Rana catesbeiana , RoscovitinaRESUMEN
The exercise pressor reflex (EPR) is generated by group III and IV muscle afferents during exercise to increase cardiovascular function. Muscle contraction is triggered by ACh, which is metabolized into choline that could serve as a signal of exercise-induced activity. We demonstrate that ACh can induce current in muscle afferents neurons isolated from male Sprague-Dawley rats. The nicotinic ACh receptors (nAChRs) appear to be expressed by some group III-IV neurons since capsaicin (TRPV1) and/or ATP (P2X) induced current in 56% of ACh-responsive neurons. α7- And α4ß2-nAChRs have been shown to be expressed in sensory neurons. An α7-nAChR antibody stained 83% of muscle afferent neurons. Functional expression was demonstrated by using the specific α7-nAChR blockers α-conotoxin ImI (IMI) and methyllycaconitine (MLA). MLA inhibited ACh responses in 100% of muscle afferent neurons, whereas IMI inhibited ACh responses in 54% of neurons. Dihydro-ß-erythroidine, an α4ß2-nAChR blocker, inhibited ACh responses in 50% of muscle afferent neurons, but recovery from block was not observed. Choline, an α7-nAChR agonist, elicited a response in 60% of ACh-responsive neurons. Finally, we demonstrated the expression of α7-nAChR by peripherin labeled (group IV) afferent fibers within gastrocnemius muscles. Some of these α7-nAChR-positive fibers were also positive for P2X3 receptors. Thus choline could serve as an activator of the EPR by opening α7-nAChR expressed by group IV (and possible group III) afferents. nAChRs could become pharmacological targets for suppressing the excessive EPR activation in patients with peripheral vascular disease.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Músculo Esquelético/inervación , Neuronas Aferentes/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Aconitina/análogos & derivados , Aconitina/farmacología , Potenciales de Acción , Animales , Capsaicina/farmacología , Neuronas Colinérgicas/efectos de los fármacos , Neuronas Colinérgicas/fisiología , Conotoxinas/farmacología , Dihidro-beta-Eritroidina/farmacología , Ganglios Espinales/citología , Masculino , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidores , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Cardiovascular adjustments to exercise are partially mediated by group III/IV (small to medium) muscle afferents comprising the exercise pressor reflex (EPR). However, this reflex can be inappropriately activated in disease states (e.g., peripheral vascular disease), leading to increased risk of myocardial infarction. Here we investigate the voltage-dependent calcium (CaV) channels expressed in small to medium muscle afferent neurons as a first step toward determining their potential role in controlling the EPR. Using specific blockers and 5 mM Ba(2+) as the charge carrier, we found the major calcium channel types to be CaV2.2 (N-type) > CaV2.1 (P/Q-type) > CaV1.2 (L-type). Surprisingly, the CaV2.3 channel (R-type) blocker SNX482 was without effect. However, R-type currents are more prominent when recorded in Ca(2+) (Liang and Elmslie 2001). We reexamined the channel types using 10 mM Ca(2+) as the charge carrier, but results were similar to those in Ba(2+). SNX482 was without effect even though â¼27% of the current was blocker insensitive. Using multiple methods, we demonstrate that CaV2.3 channels are functionally expressed in muscle afferent neurons. Finally, ATP is an important modulator of the EPR, and we examined the effect on CaV currents. ATP reduced CaV current primarily via G protein ßγ-mediated inhibition of CaV2.2 channels. We conclude that small to medium muscle afferent neurons primarily express CaV2.2 > CaV2.1 ≥ CaV2.3 > CaV1.2 channels. As with chronic pain, CaV2.2 channel blockers may be useful in controlling inappropriate activation of the EPR.
Asunto(s)
Canales de Calcio/metabolismo , Músculo Esquelético/inervación , Neuronas Aferentes/fisiología , Potenciales de Acción , Adenosina Trifosfato/farmacología , Animales , Bario/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/clasificación , Canales de Calcio/genética , Línea Celular Tumoral , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Humanos , Masculino , Músculo Esquelético/fisiología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/metabolismo , Ratas , Ratas Sprague-Dawley , ReflejoRESUMEN
Sensory neurons in the dorsal root ganglia (DRG) express a subset of voltage dependent sodium channels (NaV) including NaV1.1, 1.6, 1.7, 1.8 and 1.9. Previous work supported preferential localization of NaV1.8 channels to small-medium diameter, nociceptive afferent neurons. However, we recently published evidence that NaV1.8 was the dominant NaV channel expressed in the somas of small, medium and large diameter muscle afferent neurons, which is consistent with other reports. Here, we extend those results to show that NaV1.8 expression is not correlated with afferent neuron diameter. Using immunocytochemistry, we found NaV1.8 expression in ~50% of sensory afferent neurons with diameters ranging from 20 to 70 µm. In addition, electrophysiological analysis shows that the kinetic and inactivation properties of NaV1.8 current are invariant with neuron size. These data add further support to the idea that NaV1.8 contributes to the electrical excitability of both nociceptive and non-nociceptive sensory neurons.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.8/genética , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Animales , Tamaño de la Célula , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Masculino , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (Na(V)) channels. In patch-clamp recordings, we found that the dominant Na(V) current in the majority of identified neurons was insensitive to tetrodotoxin (TTX-R), with Na(V) current in only a few (14%) neurons showing substantial (>50%) TTX sensitivity (TTX-S). The TTX-R current was sensitive to a Na(V)1.8 channel blocker, A803467. Immunocytochemistry demonstrated labeling of muscle afferent neurons by a Na(V)1.8 antibody, which further supported expression of these channels. A portion of the TTX-R Na(V) current appeared to be noninactivating during our 25-ms voltage steps, which suggested activity of Na(V)1.9 channels. The majority of the noninactivating current was insensitive to A803467 but sensitive to extracellular sodium. Immunocytochemistry showed labeling of muscle afferent neurons by a Na(V)1.9 channel antibody, which supports expression of these channels. Further examination of the muscle afferent neurons showed that functional TTX-S channels were expressed, but were largely inactivated at physiological membrane potentials. Immunocytochemistry showed expression of the TTX-S channels Na(V)1.6 and Na(V)1.7 but not Na(V)1.1. Na(V)1.8 and Na(V)1.9 appear to be the dominant functional sodium channels in small- to medium-diameter muscle afferent neurons. The expression of these channels is consistent with the identification of these neurons as Group III and IV, which mediate the exercise pressor reflex.
Asunto(s)
Neuronas Aferentes/fisiología , Canales de Sodio Activados por Voltaje/fisiología , Potenciales de Acción , Compuestos de Anilina/farmacología , Animales , Furanos/farmacología , Ganglios Espinales/citología , Ganglios Espinales/fisiología , Expresión Génica , Masculino , Músculo Esquelético/inervación , Ratas , Ratas Sprague-Dawley , Sodio/metabolismo , Tetrodotoxina/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.
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Cisteína/análisis , Disulfuros/química , Evolución Química , Péptidos/química , Selenocisteína/química , omega-Conotoxina GVIA/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Proteolisis , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
T-type calcium channels (Ca(V)3) play an important role in many physiological and pathological processes, including cancerogenesis. Ca(V)3 channel blockers have been proposed as potential cancer treatments. Roscovitine, a trisubstituted purine, is a cyclin-dependent kinase (CDK) inhibitor that is currently undergoing phase II clinical trials as an anticancer drug and has been shown to affect calcium and potassium channel activity. Here, we investigate the effect of roscovitine on Ca(V)3.1 channels. Ca(V)3.1 channels were transiently expressed in human embryonic kidney 293 cells, and currents were recorded by using the whole-cell patch-clamp technique. Roscovitine blocks Ca(V)3.1 channels with higher affinity for depolarized cells (EC50 of 10 µM), which is associated with a negative shift in the voltage dependence of closed-state inactivation. Enhanced inactivation is mediated by roscovitine-induced acceleration of closed-state inactivation and slowed recovery from inactivation. Small effects of roscovitine were also observed on T-channel deactivation and open-state inactivation, but neither could explain the inhibitory effect. Roscovitine inhibits Ca(V)3.1 channels within the therapeutic range (10-50 µM) in part by stabilizing the closed-inactivated state. The ability of roscovitine to block multiple mediators of proliferation, including CDKs and Ca(V)3.1 channels, may facilitate its anticancer properties.
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Canales de Calcio Tipo T/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Purinas/farmacología , Potenciales de Acción/fisiología , Animales , Antineoplásicos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/fisiología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Fenómenos Electrofisiológicos/efectos de los fármacos , Fenómenos Electrofisiológicos/fisiología , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Cinética , Potenciales de la Membrana/fisiología , Ratas , Roscovitina , TransfecciónRESUMEN
Ca(V)2.2 (N-type) and Ca(V)1.2 (L-type) calcium channels gate differently in response to membrane depolarization, which is critical to the unique physiological functions mediated by these channels. We wondered if the source for these differences could be identified. As a first step, we examined the effect of domain exchange between N-type and L-type channels on activation-deactivation kinetics, which were significantly different between these channels. Kinetic analysis of chimeric channels revealed N-channel-like deactivation for all chimeric channels containing N-channel domain III, while activation appeared to be a more distributed function across domains. This led us to hypothesize that domain III was an important regulator of N-channel closing. This idea was further examined with R-roscovitine, which is a trisubstituted purine that slows N-channel deactivation by exclusively binding to activated N-channels. L-channels lack this response to roscovitine, which allowed us to use N-L chimeras to test the role of domain III in roscovitine modulation of N-channel deactivation. In support of our hypothesis, all chimeric channels containing the N-channel domain III responded to roscovitine with slowed deactivation, while those chimeric channels with L-channel domain III did not. Thus a combination of kinetic and pharmacological evidence supports the hypothesis that domain III is an important regulator of N-channel closing. Our results support specialization of gating functions among calcium channel domains.
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Fenómenos Biofísicos/fisiología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/fisiología , Activación del Canal Iónico/fisiología , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Canales de Calcio Tipo N/genética , Estimulación Eléctrica , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Proteínas Mutantes Quiméricas/genética , Técnicas de Placa-Clamp , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Purinas/farmacología , Conejos , Roscovitina , TransfecciónRESUMEN
Propofol is commonly used to induce anesthesia but has been associated with some negative cardiovascular side effects, including negative inotropy, hypotension, and bradycardia. This study investigated the effect of propofol on L-type calcium current in acutely isolated human atrial myocytes to better understand the mechanism of these side effects. After informed consent was obtained, the atrial appendage was obtained from patients undergoing open-heart surgery who required cardiopulmonary bypass. Atrial myocytes were isolated using enzymatic digestion, and L-type calcium currents were recorded using the whole-cell patch clamp technique. Propofol enhanced the magnitude and speed of voltage-dependent inactivation of L-current. As a result, the propofol-induced inhibition was increased by protocols that increased inactivation such as longer voltage step duration, holding potential depolarization, and increased pulsing frequency. The preferential enhancement of L-channel inactivation by propofol can explain the associated cardiovascular side effects. The depolarized resting potential of arterial smooth muscle may render the L-channels in these cells particularly sensitive to propofol-induced inhibition, which could explain the hypotension observed in some patients. The enhancement of both inactivation kinetics and steady-state inactivation by propofol can also explain the negative inotropic effect. However, the enhanced voltage-dependent inactivation and use dependence could have beneficial effects for patients prone to certain arrhythmias and tachycardia.
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Anestésicos Intravenosos/farmacología , Apéndice Atrial/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Propofol/farmacología , Anestésicos Intravenosos/efectos adversos , Apéndice Atrial/metabolismo , Bradicardia/inducido químicamente , Bloqueadores de los Canales de Calcio/efectos adversos , Canales de Calcio Tipo L/metabolismo , Estimulación Eléctrica , Humanos , Hipotensión/inducido químicamente , Cinética , Potenciales de la Membrana , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Propofol/efectos adversosRESUMEN
We have found that phospholemman (PLM) associates with and modulates the gating of cardiac L-type calcium channels (Wang et al., Biophys J 98: 1149-1159, 2010). The short 17 amino acid extracellular NH(2)-terminal domain of PLM contains a highly conserved PFTYD sequence that defines it as a member of the FXYD family of ion transport regulators. Although we have learned a great deal about PLM-dependent changes in calcium channel gating, little is known regarding the molecular mechanisms underlying the observed changes. Therefore, we investigated the role of the PFTYD segment in the modulation of cardiac calcium channels by individually replacing Pro-8, Phe-9, Thr-10, Tyr-11, and Asp-12 with alanine (P8A, F9A, T10A, Y11A, D12A). In addition, Asp-12 was changed to lysine (D12K) and cysteine (D12C). As expected, wild-type PLM significantly slows channel activation and deactivation and enhances voltage-dependent inactivation (VDI). We were surprised to find that amino acid substitutions at Thr-10 and Asp-12 significantly enhanced the ability of PLM to modulate Ca(V)1.2 gating. T10A exhibited a twofold enhancement of PLM-induced slowing of activation, whereas D12K and D12C dramatically enhanced PLM-induced increase of VDI. The PLM-induced slowing of channel closing was abrogated by D12A and D12C, whereas D12K and T10A failed to impact this effect. These studies demonstrate that the PFXYD motif is not necessary for the association of PLM with Ca(V)1.2. Instead, since altering the chemical and/or physical properties of the PFXYD segment alters the relative magnitudes of opposing PLM-induced effects on Ca(V)1.2 channel gating, PLM appears to play an important role in fine tuning the gating kinetics of cardiac calcium channels and likely plays an important role in shaping the cardiac action potential and regulating Ca(2+) dynamics in the heart.
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Secuencias de Aminoácidos , Sustitución de Aminoácidos , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Activación del Canal Iónico/genética , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Alanina/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Técnicas de Placa-Clamp , Fosfoproteínas/genética , Treonina/genéticaRESUMEN
N-type calcium channels play an important role in synaptic transmission and a drug that blocks these channels has become an important tool in controlling chronic pain. The development of new N-channel-targeted drugs is dependent on a better understanding of the gating of these channels and how that gating can be modulated. We have previously concluded that omega-conotoxin GVIA (GVIA) is a gating modifier that acts by destabilizing the N-channel open state. However, this conclusion was largely based on our modeling results and requires experimental support. Roscovitine, a tri-substituted purine, has been shown to stabilize the N-channel open state to slow gating charge relaxation, which provides a direct test of our hypothesis for GVIA-induced gating modification. We found that roscovitine could modulate gating current in the presence of GVIA, which shows that roscovitine can still affect the gating of the GVIA-bound N-channel. However, the magnitude of the roscovitine-induced slowing of Off-gating current was significantly reduced. In addition to confirming our hypothesis, our evidence supports an additional effect of GVIA to alter gating transitions between N-channel closed states. By strongly limiting access to the N-channel open state, GVIA analogs that selectively induce this modulation could provide the basis for the next generation drugs that treat chronic pain.
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Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , omega-Conotoxina GVIA/farmacología , Canales de Calcio Tipo N/fisiología , Células Cultivadas , Humanos , Purinas/farmacología , RoscovitinaRESUMEN
Human ether-à-go-go-related gene (HERG) potassium channels play an important role in cardiac action potential repolarization, and HERG dysfunction can cause cardiac arrhythmias. However, recent evidence suggests a role for HERG in the proliferation and progression of multiple types of cancers, making it an attractive target for cancer therapy. Ceramide is an important second messenger of the sphingolipid family, which due to its proapoptotic properties has shown promising results in animal models as an anticancer agent. Yet the acute effects of ceramide on HERG potassium channels are not known. In the present study we examined the effects of cell-permeable C(6)-ceramide on HERG potassium channels stably expressed in HEK-293 cells. C(6)-ceramide (10 microM) reversibly inhibited HERG channel current (I(HERG)) by 36 +/- 5%. Kinetically, ceramide induced a significant hyperpolarizing shift in the current-voltage relationship (DeltaV(1/2) = -8 +/- 0.5 mV) and increased the deactivation rate (43 +/- 3% for tau(fast) and 51 +/- 3% for tau(slow)). Mechanistically, ceramide recruited HERG channels within caveolin-enriched lipid rafts. Cholesterol depletion and repletion experiments and mathematical modeling studies confirmed that inhibition and gating effects are mediated by separate mechanisms. The ceramide-induced hyperpolarizing gating shift (raft mediated) could offset the impact of inhibition (raft independent) during cardiac action potential repolarization, so together they may nullify any negative impact on cardiac rhythm. Our results provide new insights into the effects of C(6)-ceramide on HERG channels and suggest that C(6)-ceramide can be a promising therapeutic for cancers that overexpress HERG.