Presence Of Nav1 Research Articles

Loss of sodium current caused by a Brugada syndrome–associated variant is determined by patient-specific genetic background

BackgroundBrugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium channel alpha subunit (NaV1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families. ObjectiveThe purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M. MethodsWe studied sodium currents from patient-specific human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases. ResultsOur results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R. ConclusionOur results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.

TTX-resistant sodium currents in medial prefrontal cortex pyramidal neurons depend on extracellular Ca2+ concentration

Background: Previous reports reported the presence of TTX-resistant Nav1.8 and Nav1.9 sodium chan­nels in the cortex pyramidal neurons. A characteristic feature of Nav1.9 channels is activation at voltages close to — 70 mV. Therefore, they do not participate directly in the action potential but contribute to the regulation of the resting membrane potential. Their physiological role is modulation of cell excitability. The aim of the study was to investigate, with the use of patch clamp technique, the dependence of the activation thresholds of TTX–resistant sodium currents on the concentration of extracellular calcium in medial prefrontal cortex pyramidal neurons in rats. Results: The recorded values of the threshold of the voltage-dependent sodium currents were in the range of -65 mV to -75 mV. This suggests that the sodium currents may result from the presence of Nav1.9 channels in the rat pyramidal neurons. The threshold for the activation of sodium currents depended on the concentration of Ca2+. Increasing the concentration of calcium ions in the extracellular solution by 5 mM caused the depolarizing shift in the activation potential by about 10 mV. The effect of calcium ion concentration on the potential of TTX-resistant currents activation suggests that Nav1.9 channels are modulated by extracellular Ca2+ concentration. Conclusions: The study has experimentally confirmed that TTX-resistant channels are present in the cell membrane of the rat prefrontal cortex pyramidal neurons and may, therefore, take a physiological role in the conductivity of sodium currents in a manner dependent on the concentration of extracellular ions.

NaV1.9 channels in muscle afferent neurons and axons.

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.

Class III antiarrhythmic drugs amiodarone and dronedarone impair KIR 2.1 backward trafficking.

Drug‐induced ion channel trafficking disturbance can cause cardiac arrhythmias. The subcellular level at which drugs interfere in trafficking pathways is largely unknown. KIR2.1 inward rectifier channels, largely responsible for the cardiac inward rectifier current (IK 1), are degraded in lysosomes. Amiodarone and dronedarone are class III antiarrhythmics. Chronic use of amiodarone, and to a lesser extent dronedarone, causes serious adverse effects to several organs and tissue types, including the heart. Both drugs have been described to interfere in the late‐endosome/lysosome system. Here we defined the potential interference in KIR2.1 backward trafficking by amiodarone and dronedarone. Both drugs inhibited IK 1 in isolated rabbit ventricular cardiomyocytes at supraclinical doses only. In HK‐KWGF cells, both drugs dose‐ and time‐dependently increased KIR2.1 expression (2.0 ± 0.2‐fold with amiodarone: 10 μM, 24 hrs; 2.3 ± 0.3‐fold with dronedarone: 5 μM, 24 hrs) and late‐endosomal/lysosomal KIR2.1 accumulation. Increased KIR2.1 expression level was also observed in the presence of Nav1.5 co‐expression. Augmented KIR2.1 protein levels and intracellular accumulation were also observed in COS‐7, END‐2, MES‐1 and EPI‐7 cells. Both drugs had no effect on Kv11.1 ion channel protein expression levels. Finally, amiodarone (73.3 ± 10.3% P < 0.05 at −120 mV, 5 μM) enhanced IKIR 2.1 upon 24‐hrs treatment, whereas dronedarone tended to increase IKIR 2.1 and it did not reach significance (43.8 ± 5.5%, P = 0.26 at −120 mV; 2 μM). We conclude that chronic amiodarone, and potentially also dronedarone, treatment can result in enhanced IK 1 by inhibiting KIR2.1 degradation.

A 3.7 kb fragment of the mouse Scn10a gene promoter directs neural crest but not placodal lineage EGFP expression in a transgenic animal.

Under physiological conditions, the voltage-gated sodium channel Nav1.8 is expressed almost exclusively in primary sensory neurons. The mechanism restricting Nav1.8 expression is not entirely clear, but we have previously described a 3.7 kb fragment of the Scn10a promoter capable of recapitulating the tissue-specific expression of Nav1.8 in transfected neurons and cell lines (Puhl and Ikeda, 2008). To validate these studies in vivo, a transgenic mouse encoding EGFP under the control of this putative sensory neuron specific promoter was generated and characterized in this study. Approximately 45% of dorsal root ganglion neurons of transgenic mice were EGFP-positive (mean diameter = 26.5 μm). The majority of EGFP-positive neurons bound isolectin B4, although a small percentage (∼10%) colabeled with markers of A-fiber neurons. EGFP expression correlated well with the presence of Nav1.8 transcript (95%), Nav1.8-immunoreactivity (70%), and TTX-R INa (100%), although not all Nav1.8-expressing neurons expressed EGFP. Several cranial sensory ganglia originating from neurogenic placodes, such as the nodose ganglion, failed to express EGFP, suggesting that additional regulatory elements dictate Scn10a expression in placodal-derived sensory neurons. EGFP was also detected in discrete brain regions of transgenic mice. Quantitative PCR and Nav1.8-immunoreactivity confirmed Nav1.8 expression in the amygdala, brainstem, globus pallidus, lateral and paraventricular hypothalamus, and olfactory tubercle. TTX-R INa recorded from EGFP-positive hypothalamic neurons demonstrate the usefulness of this transgenic line to study novel roles of Nav1.8 beyond sensory neurons. Overall, Scn10a-EGFP transgenic mice recapitulate the majority of the Nav1.8 expression pattern in neural crest-derived sensory neurons.

Specific changes in conduction velocity recovery cycles of single nociceptors in a patient with erythromelalgia with the I848T gain-of-function mutation of Nav1.7

Seven patients diagnosed with erythromelalgia (EM) were investigated by microneurography to record from unmyelinated nerve fibers in the peroneal nerve. Two patients had characterized variants of sodium channel Nav1.7 (I848T, I228M), whereas no mutations of coding regions of Navs were found in 5 patients with EM. Irrespective of Nav1.7 mutations, more than 50% of the silent nociceptors in the patients with EM showed spontaneous activity. In the patient with mutation I848T, all nociceptors, but not sympathetic efferents, displayed enhanced early subnormal conduction in the velocity recovery cycles and the expected late subnormality was reversed to supranormal conduction. The larger hyperpolarizing shift of activation might explain the difference to the I228M mutation. Sympathetic fibers that lack Nav1.8 did not show supranormal conduction in the patient carrying the I848T mutation, confirming in human subjects that the presence of Nav1.8 crucially modulates conduction in cells expressing EM mutant channels. The characteristic pattern of changes in conduction velocity observed in the patient with the I848T gain-of function mutation in Nav1.7 could be explained by axonal depolarization and concomitant inactivation of Nav1.7. If this were true, activity-dependent hyperpolarization would reverse inactivation of Nav1.7 and account for the supranormal CV. This mechanism might explain normal pain thresholds under resting conditions.

Importance of gradients in membrane properties and electrical coupling in sinoatrial node pacing.

The sinoatrial node (SAN) is heterogeneous in terms of cell size, ion channels, current densities, connexins and electrical coupling. For example, Nav1.5 (responsible for I Na) and Cx43 (responsible for electrical coupling) are absent from the centre of the SAN (normally the leading pacemaker site), but present in the periphery (at SAN-atrial muscle junction). To test whether the heterogeneity is important for the functioning of the SAN, one- and two-dimensional models of the SAN and surrounding atrial muscle were created. Normal functioning of the SAN (in terms of cycle length, position of leading pacemaker site, conduction times, activation and repolarization sequences and space constants) was observed when, from the centre to the periphery, (i) cell characteristics (cell size and ionic current densities) were changed in a gradient fashion from a central-type (lacking I Na) to a peripheral-type (possessing I Na) and (ii) coupling conductance was increased in a gradient fashion. We conclude that the heterogeneous nature of the node is important for its normal functioning. The presence of Nav1.5 and Cx43 in the periphery may be essential for the node to be able to drive the atrial muscle: Nav1.5 provides the necessary depolarizing current and Cx43 delivers it to the atrial muscle.

Expression of NaV1.9 channels by muscle afferent neurons (1129.10)

Background: The exercise pressor reflex helps regulate cardiovascular responses to exercise. The voltage‐gated sodium channel (Nav) 1.9 has unique properties that could help to generate excessive neuronal activity. We have previously demonstrated the presence of Nav1.9 channels using antibody staining of identified neurons, but were not able to demonstrate functional activity (J Neurophysiol. 108: 2230, 2012). Methods: We voltage‐clamped identified muscle afferent neurons isolated from adult male Sprague Dawley rats and used a high fluoride internal solution to help separate Nav1.9 from Nav1.8. Results: We found a Nav1.9‐like current that activated at voltages 20 mV hyperpolarized to the main Nav1.8 current in 69% of muscle afferent neurons (n=13). This current exhibited significantly slower activation (8 ± 3 ms; n=9) compared with cells that showed only Nav1.8 (4 ± 1 ms; n=4). The Nav current in cells with Nav1.9‐like current inactivated with multiple components compared with a single component inactivation of neurons that showed only Nav1.8. Unexpectedly, the size of the Nav1.9 current declined (ran down) with time during whole‐cell recording, while the Nav1.8 current level appeared to be invariant with time. The current density of the Nav1.9‐like current declined 43% ± 35% (mean ± SD, n=5) over approximately 5 min. We are investigating the role of dialyzable molecules such as ATP in the maintenance of Nav1.9 current. Conclusion: Nav1.9 channels are functionally expressed in muscle afferent neurons. The kinetic properties of Nav1.9 (e.g. slow inactivation) could make this channel an important regulator of muscle afferent activity to exacerbate the exercise pressor reflex.Grant Funding Source: NIH grant AR059397 (KSE) and ATSU BMS grant (TLM)

The Voltage-Gated Sodium Channel Nav1.8 Is Expressed in Human Sperm

The role of Na+ fluxes through voltage-gated sodium channels in the regulation of sperm cell function remains poorly understood. Previously, we reported that several genes encoding voltage-gated Na+ channels were expressed in human testis and mature spermatozoa. In this study, we analyzed the presence and function of the TTX-resistant VGSC α subunit Nav1.8 in human capacitated sperm cells. Using an RT-PCR assay, we found that the mRNA of the gene SCN10A, that encode Na v1.8, was abundantly and specifically expressed in human testis and ejaculated spermatozoa. The Na v1.8 protein was detected in capacitated sperm cells using three different specific antibodies against this channel. Positive immunoreactivity was mainly located in the neck and the principal piece of the flagellum. The presence of Na v1.8 in sperm cells was confirmed by Western blot. Functional studies demonstrated that the increases in progressive motility produced by veratridine, a voltage-gated sodium channel activator, were reduced in sperm cells preincubated with TTX (10 μM), the Na v1.8 antagonist A-803467, or a specific Na v1.8 antibody. Veratridine elicited similar percentage increases in progressive motility in sperm cells maintained in Ca2+-containing or Ca2+-free solution and did not induce hyperactivation or the acrosome reaction. Veratridine caused a rise in sperm intracellular Na+, [Na+]i, and the sustained phase of the response was inhibited in the presence of A-803467. These results verify that the Na+ channel Na v1.8 is present in human sperm cells and demonstrate that this channel participates in the regulation of sperm function.

Physiological interactions between Nav1.7 and Nav1.8 sodium channels: a computer simulation study

We have examined the question of how the level of expression of sodium channel Na(v)1.8 affects the function of dorsal root ganglion (DRG) neurons that also express Na(v)1.7 channels and, conversely, how the level of expression of sodium channel Na(v)1.7 affects the function of DRG neurons that also express Na(v)1.8, using computer simulations. Our results demonstrate several previously undescribed effects of expression of Na(v)1.7: 1) at potentials more negative than -50 mV, increasing Na(v)1.7 expression reduces current threshold. 2) Na(v)1.7 reduces, but does not eliminate, the dependence of action potential (AP) threshold on membrane potential. 3) In cells that express Na(v)1.8, the presence of Na(v)1.7 results in larger amplitude subthreshold oscillations and increases the frequency of repetitive firing. Our results also demonstrate multiple effects of expression of Na(v)1.8: 1) dependence of current threshold on membrane potential is eliminated or reversed by expression of Na(v)1.8 at ≥50% of normal values. 2) Expression of Na(v)1.8 alone, in the absence of Na(v)1.7, can support subthreshold oscillation. 3) Na(v)1.8 is required for generation of overshooting APs, and its expression results in a prolonged AP with an inflection of the falling phase. 4) Increasing levels of expression of Na(v)1.8 result in a reduction in the voltage threshold for AP generation. 5) Increasing levels of expression of Na(v)1.8 result in an attenuation of Na(v)1.7 current during activity evoked by sustained depolarization due, at least in part, to accumulation of fast inactivation by Na(v)1.7 following the first AP. These results indicate that changes in the level of expression of Na(v)1.7 and Na(v)1.8 may provide a regulatory mechanism that tunes the excitability of small DRG neurons.

G009 Regulation of the cardiac sodium channel Nav1.5 by a member of MAGUK proteins: SAP97

Nav1.5 is the voltage-gated sodium channel that initiates the cardiac action potential. A precise regulation and localisation of Nav1.5 channels in cardiomyocytes is thus necessary for correct heart function. The three last amino-acids of Nav1.5 (SIV) constitute a PDZ-domain binding motif known to interact with the syntrophin-dystrophin complex and PDZ domains found in proteins of the MAGUK family. Among their multiple roles, MAGUK proteins can cluster proteins and localize them at the plasma membrane. We investigated the interaction between SAP97, one cardiac MAGUK protein, and Nav1.5. We postulated that this interaction may be implicated in correct localisation, anchoring, turn-over and/or regulation of Nav1.5 biophysical properties. Pull-down experiments performed with Nav1.5 C-terminus fusion proteins and human or mouse heart protein extracts revealed that the association between SAP97 and Nav1.5 depends on the PDZ-domain binding motif of Nav1.5. This interaction was specific for SAP97 and Nav1.5 as no pull-down could be detected with PSD95 or ZO-1, two MAGUK proteins also expressed in human heart. The functional consequences of this interaction were studied via patch-clamp experiments. Silencing of SAP97 reduced the whole-cell sodium current measured in HEK293 cells stably expressing Nav1.5 channels without decreasing the total protein amount. In control or silenced HEK293 cells, sodium current produced by Nav1.5 δSIV channels was reduced compared to WT. Immunostainings on frozen mouse heart slices demonstrated the colocalisation of Nav1.5 and dystrophin specifically at lateral membranes, but not at the intercalated discs. The possible colocalisation of Nav1.5 and SAP97 at the level of intercalated disks is currently investigated. This would support the hypothesis of the presence of two pools of Nav1.5 channels: one targeted at lateral membranes by the syntrophin-dystrophin complex, and another one targeted at intercalated discs by SAP97. These findings strongly support the existence of an interaction between Nav1.5 and SAP97 in cardiac tissue. This interaction also depends on the presence of Nav1.5 PDZ-domain binding motif and may play a role in determining the channel density at the plasma membrane. Additional biochemistry, cytochemistry and biophysical experiments will allow us to further address this question.

Specificity, affinity and efficacy of iota-conotoxin RXIA, an agonist of voltage-gated sodium channels Na V1.2, 1.6 and 1.7

The excitotoxic conopeptide ι-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that ι-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel Na V1.6 to a more hyperpolarized level. Here, we performed voltage-clamp experiments to examine its activity against rodent Na V1.1 through Na V1.7 co-expressed with the β1 subunit in Xenopus oocytes and Na V1.8 in dissociated mouse DRG neurons. The order of sensitivity to ι-RXIA was Na V1.6 > 1.2 > 1.7, and the remaining subtypes were insensitive. The time course of ι-RXIA-activity on Na V1.6 during exposure to different peptide concentrations were well fit by single-exponential curves that provided k obs. The plot of k obs versus [ι-RXIA] was linear, consistent with a bimolecular reaction with a K d of ∼3 μM, close to the steady-state EC 50 of ∼2 μM. ι-RXIA has an unusual residue, D-Phe, and the analog with an L-Phe instead, ι-RXIA[L-Phe44], had a two-fold lower affinity and two-fold faster off-rate than ι-RXIA on Na V1.6 and furthermore was inactive on Na V1.2. ι-RXIA induced repetitive action potentials in mouse sciatic nerve with conduction velocities of both A- and C-fibers, consistent with the presence of Na V1.6 at nodes of Ranvier as well as in unmyelinated axons. Sixteen peptides homologous to ι-RXIA have been identified from a single species of Conus, so these peptides represent a rich family of novel sodium channel-targeting ligands.

Inactivation Properties of Sodium Channel Nav1.8 Maintain Action Potential Amplitude in Small DRG Neurons in the Context of Depolarization

BackgroundSmall neurons of the dorsal root ganglion (DRG) express five of the nine known voltage-gated sodium channels. Each channel has unique biophysical characteristics which determine how it contributes to the generation of action potentials (AP). To better understand how AP amplitude is maintained in nociceptive DRG neurons and their centrally projecting axons, which are subjected to depolarization within the dorsal horn, we investigated the dependence of AP amplitude on membrane potential, and how that dependence is altered by the presence or absence of sodium channel Nav1.8.ResultsIn small neurons cultured from wild type (WT) adult mouse DRG, AP amplitude decreases as the membrane potential is depolarized from -90 mV to -30 mV. The decrease in amplitude is best fit by two Boltzmann equations, having V1/2 values of -73 and -37 mV. These values are similar to the V1/2 values for steady-state fast inactivation of tetrodotoxin-sensitive (TTX-s) sodium channels, and the tetrodotoxin-resistant (TTX-r) Nav1.8 sodium channel, respectively. Addition of TTX eliminates the more hyperpolarized V1/2 component and leads to increasing AP amplitude for holding potentials of -90 to -60 mV. This increase is substantially reduced by the addition of potassium channel blockers. In neurons from Nav1.8(-/-) mice, the voltage-dependent decrease in AP amplitude is characterized by a single Boltzmann equation with a V1/2 value of -55 mV, suggesting a shift in the steady-state fast inactivation properties of TTX-s sodium channels. Transfection of Nav1.8(-/-) DRG neurons with DNA encoding Nav1.8 results in a membrane potential-dependent decrease in AP amplitude that recapitulates WT properties.ConclusionWe conclude that the presence of Nav1.8 allows AP amplitude to be maintained in DRG neurons and their centrally projecting axons even when depolarized within the dorsal horn.

Altered Ion Channels in an Animal Model of Charcot-Marie-Tooth Disease Type IA

How demyelination and remyelination affect the function of myelinated axons is a fundamental aspect of demyelinating diseases. We examined this issue in Trembler-J mice, a genetically authentic model of a dominantly inherited demyelinating neuropathy of humans. The K+ channels Kv1.1 and Kv1.2 channels were often improperly located in the paranodal axon membrane, typically associated with improperly formed paranodes, and in unmyelinated segments between internodes. As in wild-type nerves, Trembler-J nodes contained Nav1.6, ankyrin-G, betaIV-spectrin, and KCNQ2, but, unlike wild-type nerves, they also contained Kv3.1b and Nav1.8. In unmyelinated segments bordered by myelin sheaths, these proteins were clustered in heminodes and did not appear to be diffusely localized in the unmyelinated segments themselves. Nodes and heminodes were contacted by Schwann cells processes that did not have the ultrastructural or molecular characteristics of mature microvilli. Despite the presence of Nav1.8, a tetrodotoxin-resistant sodium channel, sciatic nerve conduction was at least as sensitive to tetrodotoxin in Trembler-J nerves as in wild-type nerves. Thus, the profound reorganization of axonal ion channels and the aberrant expression of novel ion channels likely contribute to the altered conduction in Trembler-J nerves.

Distribution of calcium binding proteins, sodium channels and persistent sodium current in the rat ventral respiratory group

The ventral respiratory group (VRG) is important in generating both the breathing rhythm and motor pattern. The VRG is generally subdivided into 4 regions, the Botzinger complex, preBotzinger Complex (pBc), rostral VRG, and caudal VRG based on neuron discharge patterns and axonal projections. Anatomical markers provide a potential bridge between the physiological analysis of the respiratory system and the neurochemical elements that are the ultimate building blocks of this system. An example of this approach is the coincidence of NK1 receptors with neurons in the pBc complex [1]. VRG labeling by antibodies to calcium binding proteins (parvalbumin, calbindin, and calretinin) in some respects complements the NK1 immunoreactivity. Parvalbumin has been suggested to label VRG neurons [2]. We have verified this by demonstrating that parvalbumin positive neurons project to the ipsilateral and contralateral VRG as well as to the phrenic nucleus. We have also found that a prominent gap in the column of VRG related parvalbumin cells [2] likely corresponds to the pBc since parvalbumin cells are rare in this zone and never co-localize with NK1 receptors. Calbindin and calretinin immunoreactive cells are scattered in the pBc and rostral VRG but rare in the Botzinger complex. Calbindin neurons are intermingled, but not colocalized with pBc NK1 cells. Calretinin is not colocalized with NK1, except for a small population of cells at the caudal ventral edge of the pBc, which likely corresponds to NK1 bulbospinal neurons [3]. Finally, preliminary evidence indicates glycine immunoreactivity in some parvalbumin neurons within the Botzinger complex region. In addition to this compartmentalized distribution of calcium binding proteins within the VRG, preliminary evidence is consistent with a differential distribution of Na channel alpha subunits. A slowly inactivating persistent Na current is postulated to underlie the pacemaker activity seen in a subset of pBc neurons [4]. Within the CNS at least 5 different Na channel alpha subunits have been identified, termed Nav1.1, 1.2, 1.3, 1.5 and 1.6. Of these Nav1.6 has been most strongly linked to persistent Na current. Immunohistochemical examination of Nav1.2, 1.3 and 1.6 demonstrated Nav1.2 is widely expressed in the VRG including some NK1 neurons. Nav1.3 was present in cranial motoneurons. In neurons acutely dissociated from the pBc of 1–15 day old rats, whole-cell voltage clamp recordings were used to analyze transient and persistent Na currents and single-cell RT-PCR was used to probe for Nav1.1, 1.2 and 1.6 mRNA. Whole-cell recordings were made using an external solution containing 50 mM NaCl. Slow ramp depolarization from -80 to +30 mV revealed a TTX-sensitive, persistent Na current. The current kinetics were dependent upon the ramp speed. A slow ramp (100 mV/s), elicited an inward non-inactivating current in 42% (10 of 24) of neurons sampled from the pBc. These results are consistent with a role for persistent Na current in regulation of the subthreshold behavior, including pacemaker activity. Moreover, single-cell RT-PCR revealed the presence of Nav1.6 in 40 of 72 cells (55%). The Nav1.1 subunit mRNA was present in 47 of 82 neurons (57%) and was co-expressed with Nav1.6 in 28% of cells. Preliminary findings on Nav1.2 mRNA are consistent with the immunohistochemistry with it present in 7 of 18 (38%) pBc neurons.