Magi-1 scaffolds NaV1.8 and Slack KNa channels in dorsal root ganglion neurons regulating excitability and pain.

Voltage-dependent sodium (NaV) 1.8 channels regulate action potential generation in nociceptive neurons, identifying them as putative analgesic targets. Here, we show that NaV1.8 channel plasma membrane localization, retention, and stability occur through a direct interaction with the postsynaptic density-95/discs large/zonula occludens-1-and WW domain-containing scaffold protein called membrane-associated guanylate kinase with inverted orientation (Magi)-1. The neurophysiological roles of Magi-1 are largely unknown, but we found that dorsal root ganglion (DRG)-specific knockdown of Magi-1 attenuated thermal nociception and acute inflammatory pain and produced deficits in NaV1.8 protein expression. A competing cell-penetrating peptide mimetic derived from the NaV1.8 WW binding motif decreased sodium currents, reduced NaV1.8 protein expression, and produced hypoexcitability. Remarkably, a phosphorylated variant of the very same peptide caused an opposing increase in NaV1.8 surface expression and repetitive firing. Likewise, in vivo, the peptides produced diverging effects on nocifensive behavior. Additionally, we found that Magi-1 bound to sequence like a calcium-activated potassium channel sodium-activated (Slack) potassium channels, demonstrating macrocomplexing with NaV1.8 channels. Taken together, these findings emphasize Magi-1 as an essential scaffold for ion transport in DRG neurons and a central player in pain.-Pryce, K. D., Powell, R., Agwa, D., Evely, K. M., Sheehan, G. D., Nip, A., Tomasello, D. L., Gururaj, S., Bhattacharjee, A. Magi-1 scaffolds NaV1.8 and Slack KNa channels in dorsal root ganglion neurons regulating excitability and pain.

Protein kinase A-induced internalization of Slack channels from the neuronal membrane occurs by adaptor protein-2/clathrin-mediated endocytosis.

The sodium-activated potassium (KNa) channel Kcnt1 (Slack) is abundantly expressed in nociceptor (pain-sensing) neurons of the dorsal root ganglion (DRG), where they transmit the large outward conductance IKNa and arbitrate membrane excitability. Slack channel expression at the DRG membrane is necessary for their characteristic firing accommodation during maintained stimulation, and reduced membrane channel density causes hyperexcitability. We have previously shown that in a pro-inflammatory state, a decrease in membrane channel expression leading to reduced Slack-mediated IKNa expression underlies DRG neuronal sensitization. An important component of the inflammatory milieu, PKA internalizes Slack channels from the DRG membrane, reduces IKNa, and produces DRG neuronal hyperexcitability when activated in cultured primary DRG neurons. Here, we show that this PKA-induced retrograde trafficking of Slack channels also occurs in intact spinal cord slices and that it is carried out by adaptor protein-2 (AP-2) via clathrin-mediated endocytosis. We provide mass spectrometric and biochemical evidence of an association of native neuronal AP-2 adaptor proteins with Slack channels, facilitated by a dileucine motif housed in the cytoplasmic Slack C terminus that binds AP-2. By creating a competitive peptide blocker of AP-2-Slack binding, we demonstrated that this interaction is essential for clathrin recruitment to the DRG membrane, Slack channel endocytosis, and DRG neuronal hyperexcitability after PKA activation. Together, these findings uncover AP-2 and clathrin as players in Slack channel regulation. Given the significant role of Slack in nociceptive neuronal excitability, the AP-2 clathrin-mediated endocytosis trafficking mechanism may enable targeting of peripheral and possibly, central neuronal sensitization.

Slack KNa Channels Influence Dorsal Horn Synapses and Nociceptive Behavior.

Abstract: The sodium-activated potassium channel Slack (Kcnt1, Slo2.2) is highly expressed in dorsal root ganglion neurons where it regulates neuronal firing. Several studies have implicated the Slack channel in pain processing, but the precise mechanism or the levels within the sensory pathway where channels are involved remain unclear. Here, we furthered the behavioral characterization of Slack channel knockout mice and for the first time examined the role of Slack channels in the superficial, pain-processing lamina of the dorsal horn. We performed whole-cell recordings from spinal cord slices to examine the intrinsic and synaptic properties of putative inhibitory and excitatory lamina II interneurons. Slack channel deletion altered intrinsic properties and synaptic drive to favor an overall enhanced excitatory tone. We measured the amplitudes and paired pulse ratio of paired excitatory post-synaptic currents at primary afferent synapses evoked by electrical stimulation of the dorsal root entry zone. We found a substantial decrease in the paired pulse ratio at synapses in Slack deleted neurons compared to wildtype, indicating increased presynaptic release from primary afferents. Corroborating these data, plantar test showed Slack knockout mice have an enhanced nociceptive responsiveness to localized thermal stimuli compared to wildtype mice. Our findings suggest that Slack channels regulate synaptic transmission within the spinal cord dorsal horn and by doing so establishes the threshold for thermal nociception.

Vanadium(III)-l-cysteine enhances the sensitivity of murine breast adenocarcinoma cells to cyclophosphamide by promoting apoptosis and blocking angiogenesis.

Various epidemiological and preclinical studies have already established the cancer chemopreventive potential of vanadium-based compounds. In addition to its preventive efficacy, studies have also indicated the abilities of vanadium-based compounds to induce cell death selectively toward malignant cells. Therefore, the objective of the present investigation is to improve the therapeutic efficacy and toxicity profile of an alkylating agent, cyclophosphamide, by the concurrent use of an organovanadium complex, vanadium(III)-l-cysteine. In this study, vanadium(III)-l-cysteine (1 mg/kg body weight, per os) was administered alone as well as in combination with cyclophosphamide (25 mg/kg body weight, intraperitoneal) in concomitant and pretreatment schedule in mice bearing breast adenocarcinoma cells. The results showed that the combination treatment significantly decreased the tumor burden and enhanced survivability of tumor-bearing mice through generation of reactive oxygen species in tumor cells. These ultimately led to DNA damage, depolarization of mitochondrial membrane potential, and apoptosis in tumor cells. Further insight into the molecular pathway disclosed that the combination treatment caused upregulation of p53 and Bax and suppression of Bcl-2 followed by the activation of caspase cascade and poly (ADP-ribose) polymerase cleavage. Administration of vanadium(III)-l-cysteine also resulted in significant attenuation of peritoneal vasculature and sprouting of the blood vessels by decreasing the levels of vascular endothelial growth factor A and matrix metalloproteinase 9 in the ascites fluid of tumor-bearing mice. Furthermore, vanadium(III)-l-cysteine significantly attenuated cyclophosphamide-induced hematopoietic, hepatic, and genetic damages and provided additional survival advantages. Hence, this study suggested that vanadium(III)-l-cysteine may offer potential therapeutic benefit in combination with cyclophosphamide by augmenting anticancer efficacy and diminishing toxicity to the host.

Chemoprotective and chemosensitizing properties of selenium nanoparticle (Nano-Se) during adjuvant therapy with cyclophosphamide in tumor-bearing mice.

Cyclophosphamide (CP) is one of the widely used anticancer agents; however, it has serious deleterious effects on normal host cells due to its nonspecific action. The essential trace element Selenium (Se) is suggested to have chemopreventive and chemotherapeutic efficacy and currently used in pharmaceutical formulations. Previous report had shown Nano-Se could protect CP-induced hepatotoxicity and genotoxicity in normal Swiss albino mice; however, its role in cancer management is still not clear. The aim of present study is to investigate the chemoprotective efficacy of Nano-Se against CP-induced toxicity as well as its chemoenhancing capability when used along with CP in Swiss albino mice against Ehrlich's ascites carcinoma (EAC) cells. CP was administered (25 mg/kg b.w., i.p.) and Nano-Se was given (2 mg Se/kg b.w., p.o.) in concomitant and pretreatment schedule. Increase levels of serum hepatic marker, hepatic lipid peroxidation, DNA damage, and chromosomal aberration in CP-treated mice were significantly (P < 0.05) reversed by Nano-Se. The lowered status of various antioxidant enzymes in tumor-bearing mice after CP treatment was also effectively increased by Nano-Se. Administration of Nano-Se along with CP caused a significant reduction in tumor volume, packed cell volume, viable tumor cell count, and increased the survivability of the tumor-bearing hosts. The results suggest that Nano-Se exhibits significant antitumor and antioxidant effects in EAC-bearing mice. The potential for Nano-Se to ameliorate the CP-evoked toxicity as well as to improve the chemotherapeutic effect could have beneficial implications for patients undergoing chemotherapy with CP.

An oxovanadium(IV) complex protects murine bone marrow cells against cisplatin-induced myelotoxicity and DNA damage.

Cisplatin (CDDP) is one of the first-line anticancer drugs that has gained widespread use against various forms of human malignancies. But, the therapeutic outcome of CDDP therapy is limited due to its adverse effects including myelotoxicity and DNA damage which may lead to the subsequent risk of developing secondary cancer. Hence, in search of a suitable cytoprotectant, this study investigated the probable protective efficacy of an oxovanadium(IV) complex, namely oxovanadium(IV)-L-cysteine methyl ester complex (VC-IV) against CDDP-induced myelosuppression and genotoxic damage in the bone marrow cells of Swiss albino mice. CDDP was administered intraperitoneally (5 mg/kg b.w.) and VC-IV was administered orally (1 mg/kg b.w.) in concomitant and 7 d pretreatment schedule. Treatment with VC-IV in CDDP-treated mice significantly (p < 0.01) enhanced bone marrow cell proliferation and inhibited cell death in the bone marrow niche indicating improvement of CDDP-induced myelotoxicity. The organovanadium compound also significantly (p < 0.01) reduced the percentage of chromosomal aberrations, the frequency of micronuclei formation, and the extent of DNA damage. The observed chemoprotective effect of VC-IV was attributed to its anti-oxidant efficacy which significantly (p < 0.01) attenuated CDDP-induced generation of free radicals, and restored (p < 0.01) the levels of oxidized and reduced glutathione. Hence, VC-IV may serve as a promising candidate for future development to decrease the deleterious effects of CDDP in the bone marrow cells of cancer patients and associated secondary complications.

Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB.

Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.

Prevention of myelosuppression and genotoxicity induced by cisplatin in murine bone marrow cells: effect of an organovanadium compound vanadium(III)-l-cysteine.

Cisplatin (CDDP) is one of the first-line anticancer drugs indicated for use against various form of human malignancies; but, the therapeutic outcome of CDDP chemotherapy is limited due to the development of myelosuppression and genotoxicity which may lead to secondary cancer. Induction of oxidative stress in normal host cells is thought to be responsible for these adverse effects. Therefore, in search of a potential chemoprotectant, an oraganovanadium compound, viz., vanadium(III)-l-cysteine (VC-III) was evaluated against CDDP-induced clastogenicity and cytotoxicity in bone marrow cells of Swiss albino mice. CDDP was administered intraperitoneally (5mg/kg body weight [b.w.]) and VC-III was given by oral gavage (1mg/kg b.w.) in concomitant and pretreatment schedule. The results showed that VC-III administration significantly (P < 0.001) enhanced cell proliferation and inhibited apoptosis in the bone marrow niche indicating recovery of CDDP-induced myelosuppression. VC-III also significantly (P < 0.001) decreased the percentage of chromosomal aberrations, the frequency of micronuclei formation and the extent of DNA damage. The observed antigenotoxic and cytoprotective effect of VC-III was attributed to its attenuation of free radicals status and restoration of oxidised and reduced glutathione levels. These results suggest that VC-III is a potential candidate for future development as a chemoprotective agent against chemotherapy-associated primary and secondary complications.

Nano-Se attenuates cyclophosphamide-induced pulmonary injury through modulation of oxidative stress and DNA damage in Swiss albino mice.

Chemotherapy is an integral part of modern day treatment regimen but anticancer drugs fail to demarcate between cancerous and normal cells thereby causing severe form of systemic toxicity. Among which pulmonary toxicity is a dreadful complication developed in cancer patients upon cyclophosphamide (CP) therapy. Oxidative stress, fibrosis, and apoptosis are the major patho-mechanisms involved in CP-induced pulmonary toxicity. In the present study, we have synthesized Nano-Se, nanotechnology-based new form of elemental selenium which has significantly lower toxicity and acceptable bioavailability. In order to meet the need of effective drugs against CP-induced adverse effects, nano selenium (Nano-Se) was tested for its possible protective efficacy on CP-induced pulmonary toxicity and bone marrow toxicity. CP intoxication resulted in structural and functional lung impairment which was revealed by massive histopathological changes. Lung injury was associated with oxidative stress/lipid peroxidation as evident by increased in reactive oxygen species, nitric oxide level, and malondialdehyde (MDA) formation with decreased in level of antioxidants such as reduced glutathione, glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, and catalase. Furthermore, CP at a dose of 25 mg/kg b.w. increased pulmonary DNA damage ('comet tail') and triggered DNA fragmentation and apoptosis in mouse bone marrow cells. On the other hand, Nano-Se at a dose of 2 mg Se/kg b.w., significantly inhibited CP-induced DNA damage in bronchoalveolar lavage cells, and decreased the apoptosis and percentage of DNA fragmentation in bone marrow cells and also antagonized the reduction of the activities of antioxidant enzymes and the increase level of MDA. Thus, our results suggest that Nano-Se in pre- and co-administration may serve as a promising preventive strategy against CP-induced pulmonary toxicity.

Attenuation of cyclophosphamide-induced pulmonary toxicity in Swiss albino mice by naphthalimide-based organoselenium compound 2-(5-selenocyanatopentyl)-benzo[de]isoquinoline 1,3-dione.

CONTEXT: The widely used antineoplastic drug cyclophosphamide causes pulmonary toxicity by inducing oxidative stress. Selenium, a dietary micronutrient, has been found to protect various organs from oxidative injuries. OBJECTIVE: This study was designed to investigate the protective efficacy of an organoselenium compound 2-(5-selenocyanato-pentyl)-benzo[de]isoquinoline 1,3-dione against cyclophosphamide-induced pulmonary toxicity in Swiss albino mice. MATERIALS AND METHODS: Cyclophosphamide (25 mg/kg b.w.) was administered intraperitoneally for 10 d and the organoselenium compound (3 mg/kg b.w.) was given by oral gavage in concomitant and pretreatment schedules. Various biochemical parameters related to oxidative stress and antioxidant enzymes along with histology of lungs were evaluated to assess the effect of the test compound. RESULTS: The oral LD50 of the test compound was more than 1000 mg/kg b.w. in Swiss albino mice. The test compound substantially ameliorated cyclophosphamide-induced pulmonary injury by reducing the levels of reactive oxygen species, reactive nitrogen species, and lipid peroxidation, respectively, by 14.88, 18.54, and 21.10% in concomitant treatment schedule and by 23.89, 35.73, and 30.76% in the pretreatment schedule as well as by restoring the level of reduced glutathione and activities of glutathione-S-transferase, superoxide dismutase, catalase, and glutathione peroxidase, respectively, by 36.88, 42.43, 38.0, 35.0, and 34.06% in the concomitant treatment schedule and by 66.02, 59.29, 57.23, 71.59, and 57.22% in the pretreatment schedule. The test compound also attenuated cyclophosphamide-induced histological alterations of lung tissue. DISCUSSION AND CONCLUSION: The test compound emerged as an efficient antioxidant protecting lungs tissue from cyclophosphamide-induced injury.

Vanadium as a chemoprotectant: effect of vanadium(III)-L-cysteine complex against cyclophosphamide-induced hepatotoxicity and genotoxicity in Swiss albino mice.

Vanadium is an essential micronutrient for living systems and has antioxidant and genoprotective property. In the present study, the protective role of an organovanadium compound vanadium(III)-L-cysteine (VC-III) was evaluated against hepatotoxicity and genotoxicity induced by cyclophosphamide (CP) (25 mg/kg b.w., i.p.) in Swiss albino mice. Treatment with VC-III (1 mg/kg b.w., p.o.) mitigated CP-induced hepatic injury as indicated by reduction in activities of alanine transaminase, aspartate transaminase, alkaline phosphatase by 1.57-, 1.58- and 1.32-fold in concomitant treatment schedule and by 1.83-, 1.77- and 1.45-fold in pretreatment schedule, respectively, and confirmed by histopathological evidences. Parallel to these changes, VC-III ameliorated CP-induced oxidative stress in liver by 1.46-, 1.26-, 1.32- and 1.42-fold in concomitant treatment group and by 1.95-, 1.40-, 1.46- and 1.73-fold in pretreatment group at the level of H2O2, superoxide, nitric oxide and lipid peroxidation, respectively. VC-III also enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and glutathione (reduced) level in mice liver by 1.46-, 1.37-, 1.29-, 1.44- and 1.45-fold in concomitant treatment schedule and by 1.64-, 1.65-, 1.42-, 1.49- and 1.57-fold in pretreatment schedule, respectively. In addition, the organovanadium compound could efficiently attenuate CP-induced chromosomal aberrations, DNA fragmentation and apoptosis in bone marrow cells and DNA damage in lymphocytes by 1.49-, 1.43-, 1.48- and 1.59-fold in concomitant treatment group and by 1.76-, 1.92-, 1.99- and 2.15-fold in pretreatment group, respectively. Thus, the present study showed that VC-III could exert protection against CP-induced hepatotoxicity and genotoxicity.

Identifying the function of the NMDA NR1 C2 subunit through its interaction with Magi-2 during inflammatory pain.

Much is understood about the structure and gating properties of NMDA receptors (NMDAR), but the function of the carboxy-terminal splice variant of the NR1 subunit, NR1 C2 has never been identified. By studying the scaffolding protein Magi-2 in animal models of inflammatory pain, we discovered how NR1 C2 protein is specifically regulated. We found that Magi-2 deficiency resulted in decreased pain behavior and a concomitant reduction in NR1 C2 protein. Magi-2 contains WW domains, domains typically found in ubiquitin ligases. We identified an atypical WW-binding domain within NR1 C2 which conferred susceptibility to Nedd4-1 ubiquitin-ligase dependent degradation. We used lipidated peptidomimetics derived from the NR1 C2 sequence and found that NR1 C2 protein levels and pain behavior can be pharmacologically targeted. The function of NR1 C2 is to give lability to a pool of NMDAR, important for pain signaling.

PKA-induced internalization of slack KNa channels produces dorsal root ganglion neuron hyperexcitability.

Inflammatory mediators through the activation of the protein kinase A (PKA) pathway sensitize primary afferent nociceptors to mechanical, thermal, and osmotic stimuli. However, it is unclear which ion conductances are responsible for PKA-induced nociceptor hyperexcitability. We have previously shown the abundant expression of Slack sodium-activated potassium (K(Na)) channels in nociceptive dorsal root ganglion (DRG) neurons. Here we show using cultured DRG neurons, that of the total potassium current, I(K), the K(Na) current is predominantly inhibited by PKA. We demonstrate that PKA modulation of K(Na) channels does not happen at the level of channel gating but arises from the internal trafficking of Slack channels from DRG membranes. Furthermore, we found that knocking down the Slack subunit by RNA interference causes a loss of firing accommodation analogous to that observed during PKA activation. Our data suggest that the change in nociceptive firing occurring during inflammation is the result of PKA-induced Slack channel trafficking.

Viral genome silencing by neuronal sirtuin 1.

Neurotropic viruses remain dormant in sensory neurons for years, but upon reactivation, they can produce multiple disease states including pain symptoms. Latent viral DNA is extrachromosomal, maintained as a circular episome bound to histones. Here, we show the regulation of an adenoviral genome by the nicotinamide adenine dinucleotide (NAD(+))-dependent histone deacetylator Sirt1 in dorsal root ganglion neurons. Pharmacological modulation of Sirt1 and Sirt1 overexpression both affected viral transgene expression. We propose that age or stress-related neuronal NAD(+) depletion may be a trigger for viral reactivation.

Na+-mediated coupling between AMPA receptors and KNa channels shapes synaptic transmission.

Na(+)-activated K(+) (K(Na)) channels are expressed in neurons and are activated by Na(+) influx through voltage-dependent channels or ionotropic receptors, yet their function remains unclear. Here we show that K(Na) channels are associated with AMPA receptors and that their activation depresses synaptic responses. Synaptic activation of K(Na) channels by Na(+) transients via AMPA receptors shapes the decay of AMPA-mediated current as well as the amplitude of the synaptic potential. Thus, the coupling between K(Na) channels and AMPA receptors by synaptically induced Na(+) transients represents an inherent negative feedback mechanism that scales down the magnitude of excitatory synaptic responses.

Thermal hyperalgesia and dynamic weight bearing share similar recovery dynamics in a sciatic nerve entrapment injury model.

Chronic constriction injuries (CCI) of the sciatic nerve are widely used nerve entrapment animal models of neuropathic pain. Two common pain behaviors observed following CCI are thermal hyperalgesia and mechanical allodynia, measured by the Hargreaves and von Frey tests, respectively. While thermal hyperalgesia tends to recover by 30 days, mechanical allodynia can persist for many more months thereafter. Consequently, mechanical allodynia has been used extensively as a measure of 'chronic pain' focusing on the circuitry changes that occur within the spinal cord. Here, using the sciatic nerve cuff variant of CCI in mice, we propose that in contrast to these evoked measures of nociceptive hypersensitivity, dynamic weight bearing provides a more clinically relevant behavioral measure for ongoing pain during nerve injury. We found that the effect of sciatic nerve cuff on the ratio of weight bearing by the injured relative to uninjured hindlimbs more closely resembled that of thermal hyperalgesia, following a trend toward recovery by 30 days. We also found an increase in the percent of body weight bearing by the contralateral paw that is not seen in the previously tested behaviors. These results demonstrate that dynamic weight bearing is a reliable measure of non-evoked neuropathic pain and suggest that thermal hyperalgesia, rather than mechanical allodynia, provides a proxy measure for nerve entrapment-induced ongoing pain.

Inhibiting endocytosis in CGRP+ nociceptors attenuates inflammatory pain-like behavior.

The advantage of locally applied anesthetics is that they are not associated with the many adverse effects, including addiction liability, of systemically administered analgesics. This therapeutic approach has two inherent pitfalls: specificity and a short duration of action. Here, we identified nociceptor endocytosis as a promising target for local, specific, and long-lasting treatment of inflammatory pain. We observed preferential expression of AP2α2, an α-subunit isoform of the AP2 complex, within CGRP+/IB4- nociceptors in rodents and in CGRP+ dorsal root ganglion neurons from a human donor. We utilized genetic and pharmacological approaches to inhibit nociceptor endocytosis demonstrating its role in the development and maintenance of acute and chronic inflammatory pain. One-time injection of an AP2 inhibitor peptide significantly reduced acute and chronic pain-like behaviors and provided prolonged analgesia. We evidenced sexually dimorphic recovery responses to this pharmacological approach highlighting the importance of sex differences in pain development and response to analgesics.

NAD+ activates KNa channels in dorsal root ganglion neurons.

Although sodium-activated potassium channels (KNa) have been suggested to shape various firing patterns in neurons, including action potential repolarization, their requirement for high concentrations of Na+ to gate conflicts with this view. We characterized KNa channels in adult rat dorsal root ganglion (DRG) neurons. Using immunohistochemistry, we found ubiquitous expression of the Slack KNa channel subunit in small-, medium-, and large-diameter DRG neurons. Basal KNa channel activity could be recorded from cell-attached patches of acutely dissociated neurons bathed in physiological saline, and yet in excised inside-out membrane patches, the Na+ EC50 for KNa channels was typically high, approximately 50 mM. In some cases, however, KNa channel activity remained considerable after initial patch excision but decreased rapidly over time. Channel activity was restored in patches with high Na+. The channel rundown after initial excision suggested that modulation of channels might be occurring through a diffusible cytoplasmic factor. Sequence analysis indicated that the Slack channel contains a putative nicotinamide adenine dinucleotide (NAD+)-binding site; accordingly, we examined the modulation of native KNa and Slack channels by NAD+. In inside-out-excised neuronal patch recordings, we found a decrease in the Na+ EC50 for KNa channels from approximately 50 to approximately 20 mM when NAD+ was included in the perfusate. NAD+ also potentiated recombinant Slack channel activity. NAD+ modulation may allow KNa channels to operate under physiologically relevant levels of intracellular Na+ and hence provides an explanation as to how KNa channel can control normal neuronal excitability.

The N-terminal domain of Slack determines the formation and trafficking of Slick/Slack heteromeric sodium-activated potassium channels.

Potassium channels activated by intracellular Na(+) ions (K(Na)) play several distinct roles in regulating the firing patterns of neurons, and, at the single channel level, their properties are quite diverse. Two known genes, Slick and Slack, encode K(Na) channels. We have now found that Slick and Slack subunits coassemble to form heteromeric channels that differ from the homomers in their unitary conductance, kinetic behavior, subcellular localization, and response to activation of protein kinase C. Heteromer formation requires the N-terminal domain of Slack-B, one of the alternative splice variants of the Slack channel. This cytoplasmic N-terminal domain of Slack-B also facilitates the localization of heteromeric K(Na) channels to the plasma membrane. Immunocytochemical studies indicate that Slick and Slack-B subunits are coexpressed in many central neurons. Our findings provide a molecular explanation for some of the diversity in reported properties of neuronal K(Na) channels.

Naphthalimide based novel organoselenocyanates: finding less toxic forms of selenium that would retain protective efficacy.

A series of naphthalimide based organoselenocyanates were synthesized and screened for their toxicity as well as their ability to modulate several detoxifying/antioxidative enzyme levels at a primary screening dose of 3 mg/kg b.w. in normal Swiss albino mice for 30 days. Compound 4d showed highest activity in elevating the detoxifying/antioxidant enzymes levels.