Yukiko Muroi

Local Anesthetics Disrupt Energetic Coupling between the Voltage-sensing Segments of a Sodium Channel

Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence–voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

Molecular determinants of coupling between the domain III voltage sensor and pore of a sodium channel

In a voltage-dependent sodium channel, the activation of voltage sensors upon depolarization leads to the opening of the pore gates. To elucidate the principles underlying this conformational coupling, we investigated a putative gating interface in domain III of the sodium channel using voltage-clamp fluorimetry and tryptophan-scanning mutagenesis. Most mutations have similar energetic effects on voltage-sensor activation and pore opening. However, several mutations stabilized the activated voltage sensor while concurrently destabilizing the open pore. When mapped onto a homology model of the sodium channel, most localized to hinge regions of the gating interface. Our analysis shows that these residues are involved in energetic coupling of the voltage sensor to the pore when both are in resting and when both are in activated conformations, supporting the notion that electromechanical coupling in a voltage-dependent ion channel involves the movement of rigid segments connected by elastic hinges.

Selective silencing of NaV1.7 decreases excitability and conduction in vagal sensory neurons

Non‐technical summary  Sodium channels are obligatory for the conduction of action potentials along axons. There are several different sodium channel subtypes expressed in vagal sensory neurons, and it is difficult to pharmacologically block these subtypes selectively. We used virally delivered shRNA to selectively block the production of one of the sodium channel subtypes expressed in vagal sensory neurons, namely NaV1.7, and found that by selectively inhibiting the expression of this channel the conduction of action potentials was blocked in the majority of vagal sensory neurons. This study also shows that NaV1.7 is required for the elicitation of classical vagal reflexes such as cough.

Distinct Structural Changes in the GABAA Receptor Elicited by Pentobarbital and GABA

The barbiturate pentobarbital binds to gamma-aminobutyric acid type A (GABA(A)) receptors, and this interaction plays an important role in the anesthetic action of this drug. Depending on its concentration, pentobarbital can potentiate (approximately 10-100 microM), activate (approximately 100-800 microM), or block (approximately 1-10 mM) the channel, but the mechanisms underlying these three distinct actions are poorly understood. To investigate the drug-induced structural rearrangements in the GABA(A) receptor, we labeled cysteine mutant receptors expressed in Xenopus oocytes with the sulfhydryl-reactive, environmentally sensitive fluorescent probe tetramethylrhodamine-6-maleimide (TMRM). We then used combined voltage clamp and fluorometry to monitor pentobarbital-induced channel activity and local protein movements simultaneously in real time. High concentrations of pentobarbital induced a decrease in TMRM fluorescence (F(TMRM)) of labels tethered to two residues in the extracellular domain (alpha(1)L127C and beta(2)L125C) that have been shown previously to produce an increase in F(TMRM) in response to GABA. Label at beta(2)K274C in the extracellular end of the M2 transmembrane helix reported a small but significant F(TMRM) increase during application of low modulating pentobarbital concentrations, and it showed a much greater F(TMRM) increase at higher concentrations. In contrast, GABA decreased F(TMRM) at this site. These results indicate that GABA and pentobarbital induce different structural rearrangements in the receptor, and thus activate the receptor by different mechanisms. Labels at alpha(1)L127C and beta(2)K274C change their fluorescence by substantial amounts during channel blockade by pentobarbital. In contrast, picrotoxin blockade produces no change in F(TMRM) at these sites, and the pattern of F(TMRM) signals elicited by the antagonist SR95531 differs from that produced by other antagonists. Thus, with either channel block by antagonists or activation by agonists, the structural changes in the GABA(A) receptor protein differ during transitions that are functionally equivalent.

Selective inhibition of vagal afferent nerve pathways regulating cough using Nav 1.7 shRNA silencing in guinea pig nodose ganglia.

Adeno-associated virus delivery systems and short hairpin RNA (shRNA) were used to selectively silence the voltage-gated sodium channel NaV 1.7 in the nodose ganglia of guinea pigs. The cough reflex in these animals was subsequently assessed. NaV 1.7 shRNA was delivered to the majority of nodose ganglia neurons [50-60% transfection rate determined by green fluorescent protein (GFP) gene cotransfection] and action potential conduction in the nodose vagal nerve fibers, as evaluated using an extracellular recording technique, was markedly and significantly reduced. By contrast, <5% of neurons in the jugular vagal ganglia neurons were transfected, and action potential conduction in the jugular vagal nerve fibers was unchanged. The control virus (with GFP expression) was without effect on action potential discharge and conduction in either ganglia. In vivo, NaV 1.7 silencing in the nodose ganglia nearly abolished cough evoked by mechanically probing the tracheal mucosa in anesthetized guinea pigs. Stimuli such as capsaicin and bradykinin that are known to stimulate both nodose and jugular C-fibers evoked coughing in conscious animals was unaffected by NaV 1.7 silencing in the nodose ganglia. Nodose C-fiber selective stimuli including adenosine, 2-methyl-5-HT, and ATP all failed to evoke coughing upon aerosol challenge. These results indicate that cough is independently regulated by two vagal afferent nerve subtypes in guinea pigs, with nodose Aδ fibers regulating cough evoked mechanically from the trachea and bradykinin- and capsaicin-evoked cough regulated by C-fibers arising from the jugular ganglia.

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics.

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4–S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.

Transgene expression and effective gene silencing in vagal afferent neurons in vivo using recombinant adeno-associated virus vectors

Vagal afferent fibres innervating thoracic structures such as the respiratory tract and oesophagus are diverse, comprising several subtypes of functionally distinct C‐fibres and A‐fibres. Both morphological and functional studies of these nerve subtypes would be advanced by selective, effective and long‐term transduction of vagal afferent neurons with viral vectors. Here we addressed the hypothesis that vagal sensory neurons can be transduced with adeno‐associated virus (AAV) vectors in vivo, in a manner that would be useful for morphological assessment of nerve terminals, using enhanced green fluorescent protein (eGFP), as well as for the selective knock‐down of specific genes of interest in a tissue‐selective manner. We found that a direct microinjection of AAV vectors into the vagal nodose ganglia in vivo leads to selective, effective and long‐lasting transduction of the vast majority of primary sensory vagal neurons without transduction of parasympathetic efferent neurons. The transduction of vagal neurons by pseudoserotype AAV2/8 vectors in vivo is sufficiently efficient such that it can be used to functionally silence TRPV1 gene expression using short hairpin RNA (shRNA). The eGFP encoded by AAV vectors is robustly transported to both the central and peripheral terminals of transduced vagal afferent neurons allowing for bright imaging of the nerve endings in living tissues and suitable for structure–function studies of vagal afferent nerve endings. Finally, the AAV2/8 vectors are efficiently taken up by the vagal nerve terminals in the visceral tissue and retrogradely transported to the cell body, allowing for tissue‐specific transduction.

Targeting peripheral afferent nerve terminals for cough and dyspnea.

Chronic unproductive coughing and dyspnea are symptoms that severely diminish the quality of life in a substantial proportion of the population. There are presently few if any drugs that effectively treat these symptoms. Rational drug targets for cough and dyspnea have emerged over the recent years based on developments in our understanding of the innervation of the respiratory tract. These drug targets can be subcategorized into those that target the vagal afferent nerve endings, and those that target neural activity within the CNS. This review focuses on targets presumed to be in the peripheral terminals of afferent nerves within the airways. Conceptually, the activity of peripheral afferent nerves involved with unwanted urge-to-cough or dyspnea sensations can be inhibited by limiting the intensity of the stimulus, inhibiting the amplitude of the stimulus-induced generator potential, or inhibiting the transduction between the generator potential and action potential discharge and conduction. These mechanisms reveal many therapeutic strategies for anti-tussive and anti-dyspnea drug development with peripheral sites of action.

Targeting Voltage Gated Sodium Channels NaV1.7, NaV1.8, and NaV1.9 for Treatment of Pathological Cough

Abstract Recent advances in our understanding of voltage-gated sodium channels (NaVs) lead to the rational hypothesis that drugs capable of selective blockade of NaV subtypes may be a safe and effective strategy for the treatment of unwanted cough. Among the nine NaV subtypes (NaV1.1–NaV1.9), the afferent nerves involved in initiating cough, in common with nociceptive neurons in the somatosensory system, express mainly NaV1.7, NaV1.8, and NaV1.9. Although knowledge about the effect of selectively blocking these channels on the cough reflex is limited, their biophysical properties indicate that each may contribute to the hypertussive and allotussive state that typifies subacute and chronic nonproductive cough.

Molecular Link Between Voltage-Sensor Modification and Local Anesthetic Block

Sodium channels are a major target for many toxins and drugs including local anesthetics (LA). Gating current (Sheets and Hanck, J. Gen. Physiol.; 121(2), 2003) and fluorescence measurements (Muroi and Chanda, J. Gen. Physiol.; 133(1), 2009) show that LA binding to the pore mainly stabilizes the voltage-sensor of domains III of sodium channel in an upward (activated) conformation. The half maximal (V1/2) of the fluorescence-voltage (F-V) curves of probes attached to the voltage-sensor of domain III are left shifted by as much as 50 mV upon LA binding. To address the molecular basis of stabilization of the activated S4 upon LA binding, we systematically introduced tryptophan (or alanine) residues in the S4-S5 linker, N-terminal of S5 and C-terminal of S6 of the domain III muscle sodium channel. We examined the effect of these substitutions by voltage-clamp fluorimetry and by gating current measurements. Mutations of specific residues on the S4-S5 linker and C-terminal of the S6 segment significantly reduced or eliminated the shifts in the F-V curve upon LA binding. Furthermore, the total gating charge in the presence of LA remained unchanged in these mutants. These findings may provide insight into the structure and interactions of intermediate states during the sodium channel gating process.Support: National Institutes of Health, AHA and Shaw Scientific Award