Multimodal microwheel swarms for targeting in three-dimensional networks.

Microscale bots intended for targeted drug delivery must move through three-dimensional (3D) environments that include bifurcations, inclined surfaces, and curvature. In previous studies, we have shown that magnetically actuated colloidal microwheels (µwheels) reversibly assembled from superparamagnetic beads can translate rapidly and be readily directed. Here we show that, at high concentrations, µwheels assemble into swarms that, depending on applied magnetic field actuation patterns, can be designed to transport cargo, climb steep inclines, spread over large areas, or provide mechanical action. We test the ability of these multimodal swarms to navigate through complex, inclined microenvironments by characterizing the translation and dispersion of individual µwheels and swarms of µwheels on steeply inclined and flat surfaces. Swarms are then studied within branching 3D vascular models with multiple turns where good targeting efficiencies are achieved over centimeter length scales. With this approach, we present a readily reconfigurable swarm platform capable of navigating through 3D microenvironments.

Characterization and Optimization of the Novel Transient Receptor Potential Melastatin 2 Antagonist tatM2NX.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable channel activated by adenosine diphosphate ribose metabolites and oxidative stress. TRPM2 contributes to neuronal injury in the brain caused by stroke and cardiac arrest among other diseases including pain, inflammation, and cancer. However, the lack of specific inhibitors hinders the study of TRPM2 in brain pathophysiology. Here, we present the design of a novel TRPM2 antagonist, tatM2NX, which prevents ligand binding and TRPM2 activation. We used mutagenesis of tatM2NX to determine the structure-activity relationship and antagonistic mechanism on TRPM2 using whole-cell patch clamp and Calcium imaging in human embryonic kidney 293 cells with stable human TRPM2 expression. We show that tatM2NX inhibits over 90% of TRPM2 channel currents at concentrations as low as 2 µM. Moreover, tatM2NX is a potent antagonist with an IC50 of 396 nM. Our results from tatM2NX mutagenesis indicate that specific residues within the tatM2NX C terminus are required to confer antagonism on TRPM2. Therefore, the peptide tatM2NX represents a new tool for the study of TRPM2 function in cell biology and enhances our understanding of TRPM2 in disease. SIGNIFICANCE STATEMENT: TatM2NX is a potent TRPM2 channel antagonist with the potential for clinical benefit in neurological diseases. This study characterizes interactions of tatM2NX with TRPM2 and the mechanism of action using structure-activity analysis.

Single dose of 17ß-estradiol provides transient neuroprotection in female juvenile mice after cardiac-arrest and cardiopulmonary resuscitation.

Each year there are approximately 7000 out of hospital cardiac arrests in the pediatric population, with 30% resuscitation rate and a 6-10% rate of survival to hospital discharge. Survivors of cardiac arrest exhibit learning and memory deficits that are devastating during the school years. Delayed neuronal cell death occurs in the hippocampus following cardiac arrest and likely contributes to memory impairments. Circulating endogenous estrogen in young adult females has been shown to provide protection against ischemic cell death, as does chronic exogenous administration of 17ß-estradiol (E2). Chronic estrogen benefit can have undesirable feminizing effects, particularly in pre-adolescents. Here, we tested if a single-dose of E2 is neuroprotective in our pediatric cardiac arrest mouse model performed in juvenile mice. We subjected P21P25 C57Blk6 male and female mice to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). This developmental stage preceded the hormonal onset and serum estradiol and testosterone levels were not different in males and females. A single dose of E2 (100µg/kg) or vehicle was administered 30 min after resuscitation. Neuronal cell death measured 3 days after CA/CPR showed reduced hippocampal cell death in E2-treated females, but not males. Benefit of E2 in females was blocked by the P38 MAPK inhibitor, SB203580. Hippocampal-dependent memory function was equally impaired in E2-and vehicle-treated females measured in the contextual fear conditioning task at 7 days. Our findings demonstrate female-specific transient neuroprotection with E2 that does not provide sustained functional benefit.

Therapeutic hypothermia protects against ischemia-induced impairment of synaptic plasticity following juvenile cardiac arrest in sex-dependent manner.

Pediatric cardiac arrest (CA) often leads to poor neurologic outcomes, including deficits in learning and memory. The only approved treatment for CA is therapeutic hypothermia, although its utility in the pediatric population remains unclear. This study analyzed the effect of mild therapeutic hypothermia after CA in juvenile mice on hippocampal neuronal injury and the cellular model of learning and memory, termed long-term potentiation (LTP). Juvenile mice were subjected to cardiac arrest and cardiopulmonary resuscitation (CA/CPR) followed by normothermia (37°C) and hypothermia (30°C, 32°C). Histological injury of hippocampal CA1 neurons was performed 3days after resuscitation using hematoxylin and eosin (H&E) staining. Field excitatory post-synaptic potentials (fEPSPs) were recorded from acute hippocampal slices 7days after CA/CPR to determine LTP. Synaptic function was impaired 7days after CA/CPR. Mice exposed to hypothermia showed equivalent neuroprotection, but exhibited sexually dimorphic protection against ischemia-induced impairment of LTP. Hypothermia (32°C) protects synaptic plasticity more effectively in females, with males requiring a deeper level of hypothermia (30°C) for equivalent protection. In conclusion, male and female juvenile mice exhibit equivalent neuronal injury following CA/CPR and hypothermia protects both males and females. We made the surprising finding that juvenile mice have a sexually dimorphic response to mild therapeutic hypothermia protection of synaptic function, where males may need a deeper level of hypothermia for equivalent synaptic protection.

Surface-enabled propulsion and control of colloidal microwheels.

Propulsion at the microscale requires unique strategies such as the undulating or rotating filaments that microorganisms have evolved to swim. These features however can be difficult to artificially replicate and control, limiting the ability to actuate and direct engineered microdevices to targeted locations within practical timeframes. An alternative propulsion strategy to swimming is rolling. Here we report that low-strength magnetic fields can reversibly assemble wheel-shaped devices in situ from individual colloidal building blocks and also drive, rotate and direct them along surfaces at velocities faster than most other microscale propulsion schemes. By varying spin frequency and angle relative to the surface, we demonstrate that microwheels can be directed rapidly and precisely along user-defined paths. Such in situ assembly of readily modified colloidal devices capable of targeted movements provides a practical transport and delivery tool for microscale applications, especially those in complex or tortuous geometries.

Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia.

Motor deficits are present in cardiac arrest survivors and injury to cerebellar Purkinje cells (PCs) likely contribute to impairments in motor coordination and post-hypoxic myoclonus. N-Methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity is a well-established mechanism of cell death in several brain regions, but the role of NMDA receptors in PC injury remains understudied. Emerging data in cortical and hippocampal neurons indicate that the GluN2A-containing NMDA receptors signal to improve cell survival and GluN2B-containing receptors contribute to neuronal injury. This study compared neuronal injury in the hippocampal CA1 region to that in PCs and investigated the role of NMDA receptors in PC injury in our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Analysis of cell density demonstrated a 24% loss of PCs within 24 h after 8 min CA/CPR and injury stabilized to 33% by 7 days. The subunit promiscuous NMDA receptor antagonist MK-801 protected both CA1 neurons and PCs from ischemic injury following CA/CPR, demonstrating a role for NMDA receptor activation in injury to both brain regions. In contrast, the GluN2B antagonist, Co 101244, had no effect on PC loss while protecting against injury in the CA1 region. These data indicate that ischemic injury to cerebellar PCs progresses via different cell death mechanisms compared to hippocampal CA1 neurons.

Increasing small conductance Ca2+-activated potassium channel activity reverses ischemia-induced impairment of long-term potentiation.

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

A novel mouse model of pediatric cardiac arrest and cardiopulmonary resuscitation reveals age-dependent neuronal sensitivities to ischemic injury.

BACKGROUND: Pediatric sudden cardiac arrest (CA) is an unfortunate and devastating condition, often leading to poor neurologic outcomes. However, little experimental data on the pathophysiology of pediatric CA is currently available due to the scarcity of animal models. NEW METHOD: We developed a novel experimental model of pediatric cardiac arrest and cardiopulmonary resuscitation (CA/CPR) using postnatal day 20-25 mice. Adult (8-12 weeks) and pediatric (P20-25) mice were subjected to 6min CA/CPR. Hippocampal CA1 and striatal neuronal injury were quantified 3 days after resuscitation by hematoxylin and eosin (H&E) and Fluoro-Jade B staining, respectively. RESULTS: Pediatric mice exhibited less neuronal injury in both CA1 hippocampal and striatal neurons compared to adult mice. Increasing ischemia time to 8 min CA/CPR resulted in an increase in hippocampal injury in pediatric mice, resulting in similar damage in adult and pediatric brains. In contrast, striatal injury in the pediatric brain following 6 or 8 min CA/CPR remained extremely low. As observed in adult mice, cardiac arrest causes delayed neuronal death in pediatric mice, with hippocampal CA1 neuronal damage maturing at 72 h after insult. Finally, mild therapeutic hypothermia reduced hippocampal CA1 neuronal injury after pediatric CA/CPR. COMPARISON WITH EXISTING METHOD: This is the first report of a cardiac arrest and CPR model of global cerebral ischemia in mice. CONCLUSIONS: Therefore, the mouse pediatric CA/CPR model we developed is unique and will provide an important new tool to the research community for the study of pediatric brain injury.

Sexually dimorphic response of TRPM2 inhibition following cardiac arrest-induced global cerebral ischemia in mice.

Transient global cerebral ischemia due to cardiac arrest followed by resuscitation (CA/CPR) causes significant neurological damage in vulnerable neuron populations within the brain, such as hippocampal CA1 neurons. In recent years, we have implicated the transient receptor potential M2 (TRPM2) channel as a mediator of ischemic injury to neurons. We previously demonstrated that genetic and pharmacological strategies that reduce TRPM2 function preferentially protect male neurons in vitro and reduce infarct volume following experimental stroke. Due to the narrow therapeutic window for intervention following ischemic stroke, it is important to assess the role of TRPM2 in other models of cerebral ischemia. Therefore, this study utilized a modified mouse model of CA/CPR to mimic more accurately the clinical condition by maintaining body and head temperatures near the physiological range throughout. Here, we report that inhibition of TRPM2 activity with clotrimazole reduces hippocampal CA1 neuronal injury when administered 30 min after resuscitation from cardiac arrest. Consistent with our previous observations, neuroprotection was observed in male mice and no effect on injury was observed in the female. These findings provide further evidence for TRPM2 as a target for protection against cerebral ischemia in the male brain.

TRPM2 channel activation following in vitro ischemia contributes to male hippocampal cell death.

Hippocampal CA1 neurons are particularly sensitive to ischemic damage, such as experienced following cardiac arrest and cardiopulmonary resuscitation. In recent years transient receptor potential M2 (TRPM2) channels have been identified as mediators of ischemic damage. We previously demonstrated that neuroprotective strategies targeting TRPM2 channels preferentially protect male cortical neurons from ischemic injury both in vitro and in vivo. It is important to determine the role of TRPM2 in ischemic injury of hippocampal neurons as this population of neurons are particularly sensitive to ischemic injury and are therapeutic targets. Here we report significantly decreased neuronal cell death following in vitro ischemia preferentially in male hippocampal neurons using TRPM2 inhibitors or knockdown of TRPM2 expression. Electrophysiological characterization of sex-stratified cultures shows similar levels of functional TRPM2 channel expression in male and female hippocampal neurons under basal conditions. In contrast, recordings made during reperfusion following in vitro ischemia revealed that TRPM2 channels are activated only in male neurons, resulting in rapid and complete depolarization. These findings provide strong evidence for TRPM2 as a target for protection against cerebral ischemia in male brain and helps define a molecular cell death pathway that is differentially engaged in male and female neurons.

Neuroprotection of sex steroids.

Sex steroids are essential for reproduction and development in animals and humans, and sex steroids also play an important role in neuroprotection following brain injury. New data indicate that sex-specific responses to brain injury occur at the cellular and molecular levels. This review summarizes the current understanding of neuroprotection by sex steroids, particularly estrogen, androgen, and progesterone, based on both in vitro and in vivo studies. Better understanding of the role of sex steroids under physiological and pathological conditions will help us to develop novel effective therapeutic strategies for brain injury.

Isoflurane preconditioning neuroprotection in experimental focal stroke is androgen-dependent in male mice.

Isoflurane preconditioning neuroprotection in experimental stroke is male-specific. The role of androgens in the ischemic sensitivity of isoflurane preconditioned male brain and whether androgen effects are androgen receptor dependent were assessed. Male C57BL/6 mice were implanted with flutamide (androgen receptor antagonist), or castrated and implanted with testosterone, dihydrotestosterone, flutamide, letrozole (aromatase inhibitor), or vehicle 7-13 days before preconditioning. P450 estrogen aromatase wild-type and knockout mice were also evaluated. All mice were preconditioned for 4 h with 0% (sham preconditioning) or 1% isoflurane (isoflurane preconditioning) and recovered for 24 h. Mice then underwent 2 h of middle cerebral artery occlusion and were evaluated 22 h later for infarct volume. For neurobehavioral outcomes, sham and isoflurane preconditioned castrated male+/-dihydrotestosterone groups underwent 1 h of middle cerebral artery occlusion followed by 9 days of reperfusion. Isoflurane preconditioning neuroprotection relative to infarct volume outcomes were testosterone and dihydrotestosterone dose-specific and androgen receptor-dependent. Relative to long-term neurobehavioral outcomes, front paw sensorimotor function improved in isoflurane preconditioned mice regardless of androgen status while androgen replacement independently improved sensorimotor function. In contrast, isoflurane preconditioning improved cognitive function in castrates lacking endogenous androgens, but this improvement was absent in androgen replaced mice. Our findings suggest that androgen availability during isoflurane preconditioning may influence infarct volume and neurobehavioral outcomes in male mice following experimental stroke.

Poly (ADP-ribose) polymerase-1 initiated neuronal cell death pathway--do androgens matter?

Activation of poly (ADP-ribose) polymerases (PARP) contributes to ischemic damage by causing neuronal nicotinamide adenine dinucleotide (NAD(+)) depletion, release of apoptosis-inducing factor and consequent caspase-independent cell death. PARP-mediated cell death is sexually dimorphic, participating in ischemic damage in the male brain, but not the female brain. We tested the hypothesis that androgen signaling is required for this male-specific neuronal cell death pathway. We observed smaller damage following focal cerebral ischemia (MCAO) in male PARP-1 knockout mice compared to wild type (WT) as well as decreased damage in male mice treated with the PARP inhibitor PJ34. Protection from ischemic damage provided by PJ-34 in WT mice is lost after removal of testicular androgens (CAST) and rescued by androgen replacement. CAST PARP-1 KO mice exhibit increased damage compared to intact male KO mice, an effect reversed by androgen replacement in an androgen receptor-dependent manner. Lastly, we observed that ischemia causes an increase in PARP-1 expression that is diminished in the absence of testicular androgens. Our data indicate that PARP-mediated neuronal cell death in the male brain requires intact androgen-androgen receptor signaling.

Mechanism of progesterone neuroprotection of rat cerebellar Purkinje cells following oxygen-glucose deprivation.

The survival of rat Purkinje cell (PCs) cerebellar cultures was used to test the hypothesis that progesterone is protective against oxygen-glucose deprivation through potentiation of GABA(A) receptor activity. Electrophysiological recordings confirm that PCs develop robust excitatory and inhibitory synapses in culture. Exposure of cultured PCs to increasing concentrations of progesterone during oxygen-glucose deprivation revealed a concentration-dependent protection by progesterone, with significant protection observed at physiological concentrations, as low as 10 nm. The concurrent application of the GABA(A) receptor antagonist picrotoxin (100 microm) completely abolished the neuroprotection afforded by progesterone, indicating that progesterone is neuroprotective through activation of GABA(A) receptors. Progesterone potentiates GABA(A) receptor activity indirectly through its metabolites, such as allopregnanolone (ALLO). Therefore, ALLO was applied to PC cultures and was observed to produce significant protection at all concentrations tested, from 10 to 1000 nm. Finally, the inhibition of progesterone metabolism with finasteride abolished the protection afforded by progesterone without having any effect on the neuroprotection caused by ALLO. These data indicate that progesterone protects cerebellar PCs at physiological concentrations through a GABA-active metabolite.

Hydrogen-peroxide-induced toxicity of rat striatal neurones involves activation of a non-selective cation channel.

Striatal neurones are particularly vulnerable to hypoxia/ischaemia-induced damage, and free radicals are thought to be prime mediators of this neuronal destruction. It has been shown that hydrogen peroxide (H2O2), through the production of free radicals, induces rat insulinoma cell death by activation of a non-selective cation channel, which leads to irreversible cell depolarization and unregulated Ca2+ entry into the cell. In the study presented here, we demonstrate that a subpopulation of striatal neurones (medium spiny neurones) is depolarized by H2O2 through the production of free radicals. Cell-attached recordings from rat cultured striatal neurones demonstrate that exposure to H2O2 opens a large-conductance channel that is characterized by extremely long open times (seconds). Inside-out recordings show that cytoplasmically applied beta-nicotinamide adenine dinucleotide activates a channel with little voltage dependence, a linear current-voltage relationship and a single-channel conductance of between 70 and 90 pS. This channel is permeable to Na+, K+ and Ca2+ ions. Fura-2 imaging from cultured striatal neurones reveals that H2O2 exposure induces a biphasic intracellular Ca2+ increase in a subpopulation of neurones, the second, later phase resulting in Ca2+ overload. This later component of the Ca2+ response is dependent on the presence of extracellular Ca2+ and is independent of synaptic activity or voltage-gated Ca2+ channel opening. Consequently, this channel may be an important contributor to free radical-induced selective striatal neurone destruction. These results are remarkably similar to those observed for insulinoma cells and suggest that this family of non-selective cation channels has a widespread distribution in mammalian tissues.

Small-conductance calcium-activated potassium currents in mouse hyperexcitable denervated skeletal muscle.

1. Hyperexcitability in denervated skeletal muscle is associated with the expression of SK3, a small-conductance Ca2+-activated K+ channel (SK channel). SK currents were examined in dissociated fibres from flexor digitorum brevis (FDB) muscle using the whole-cell patch clamp configuration. 2. Depolarization activated a K+-selective, apamin-sensitive and iberiotoxin-insensitive current, detected as a tail current upon repolarization, in fibres from denervated but not innervated muscle. Dialysis of the fibres with 20 mM EGTA in the patch pipette solution eliminated the tail current, consistent with this current reflecting Ca2+-activated SK channels expressed only in denervated muscle. 3. Activation of SK tail currents depended on the duration of the depolarizing pulse, consistent with a rise in intracellular Ca2+ due to release from the sarcoplasmic reticulum (SR) and influx through voltage-gated Ca2+ channels. 4. The envelope of SK tail currents was diminished by 10 microM ryanodine for all pulse durations, whereas 2 mM cobalt reduced the SK tail current for pulses greater than 80 ms, demonstrating that Ca2+ release from the SR during short pulses primarily activated SK channels. 5. In current clamp mode with the resting membrane potential set at -70 mV, denervation decreased the action potential threshold by approximately 8 mV. Application of apamin increased the action potential threshold in denervated fibres to that measured in innervated fibres, suggesting that SK channel activity modulates the apparent action potential threshold. 6. These results are consistent with a model in which SK channel activity in the T-tubules of denervated skeletal muscle causes a local increase in K+ concentration that results in hyperexcitability.

Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line.

Fura-2 fluorescence was used to investigate the effects of H2O2 on [Ca2+]i in the insulin-secreting cell line CRI-G1. H2O2 (1-10 mM) caused a biphasic increase in free [Ca2+]i, an initial rise observed within 3 min and a second, much larger rise following a 30-min exposure. Extracellular calcium removal blocked the late, but not the initial, rise in [Ca2+]i. Thapsigargin did not affect either response to H2O2, but activated capacitive calcium entry, an action abolished by 10 microM La3+. Simultaneous recordings of membrane potential and [Ca2+]i demonstrated the same biphasic [Ca2+]i response to H2O2 and showed that the late increase in [Ca2+]i coincided temporally with cell membrane potential collapse. Buffering Ca2+i to low nanomolar levels prevented both phases of increased [Ca2+]i and the H2O2-induced depolarization. The H2O2-induced late rise in [Ca2+]i was prevented by extracellular application of 100 microM La3+. La3+ (100 microM) inhibited the H2O2-induced cation current and NAD-activated cation (NSNAD) channel activity in these cells. H2O2 increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD]. These data suggest that H2O2 increases [NAD], which, coupled with increased [Ca2+]i, activates NSNAD channels, causing unregulated Ca2+ entry and consequent cell death.

Reduced glutathione inhibits beta-NAD+-activated non-selective cation currents in the CRI-G1 rat insulin-secreting cell line.

1. Whole-cell voltage-clamp recordings were used to study the characteristics of a non-selective cation current, activated by intracellular beta-NAD+, present in CRI-G1 insulin-secreting cells. The monovalent cations Na+, K+ and Cs+ were equally permeant through this channel. 2. The magnitude of the beta-NAD+ current was dependent on the concentration of both beta-NAD+ and Ca2+ in the cell. The properties of the beta-NAD+-activated macroscopic current are similar to those of the beta-NAD+-activated non-selective cation channel (NSNAD) examined at the single channel level in this cell line. 3. The presence of intracellular reduced glutathione (GSH) inhibited the beta-NAD+-activated macroscopic current and the activity of the NSNAD channel in inside-out patch recordings. 4. The inhibition of beta-NAD+-activated currents by GSH is mimicked by its analogue ophthalmic acid but not by another thiol reducing agent dithiothreitol, indicating the presence of a specific GSH binding site present on the NSNAD channel or associated protein.

Leptin activates ATP-sensitive potassium channels in the rat insulin-secreting cell line, CRI-G1.

1. Whole-cell current-clamp recordings demonstrate that leptin (0.3-10 nm) hyperpolarizes CRI-G1 insulin-secreting cells. This effect is slow on onset and is not reversed on washout of the leptin. 2. Voltage-clamp recordings indicate that leptin activates a potassium conductance in the presence of intracellular ATP (5 mm), but has not effect in its absence. Following activation of ATP-sensitive K+ (KATP) current by diazoxide (0.2 mm), addition of leptin did not alter cell membrane potential or potassium current further. 3. The leptin-induced hyperpolarization and increased potassium conductance are completely inhibited by the application of the sulphonylureas tolbutamide (100 microM) and glibenclamide (0.5 microM). 4. Cell-attached and inside-out single-channel recordings indicate that leptin activates tolbutamide-sensitive KATP channels in CRI-G1 insulin-secreting cells.

Characterization of a nicotinamide-adenine dinucleotide-dependent cation channel in the CRI-G1 rat insulinoma cell line.

1. Cell-free excised membrane patches were used to examine the properties of a novel nicotinamide-adenine dinucleotide (beta-NAD+)-activated ion channel in the rat insulin-secreting cell line, CRI-G1. 2. In inside-out recordings, beta-NAD+ (0.05-1.0 mM) induced the appearance of a channel characterized by extremely slow kinetics, with mean open times in the range of seconds. The estimated EC50 for activation was 114 microM. Channel activity declined with time (run-down) following activation by beta-NAD+ in excised patches and this was not prevented by intracellular application of trypsin. 3. The single channel current-voltage relationship was linear with a conductance of 74 pS in symmetrical NaCl. The channel appears equally permeable to Na+, K+ and Cs+, exhibits an appreciable permeability to Ca2+, Mg2+ and Ba2+, but excludes anions. 4. The channel displays an unusual voltage sensitivity, with an abrupt increase in open-state probability at depolarized voltages. 5. Channel opening, in the presence of beta-NAD+, required both Ca2+ and Mg2+ to be present at the internal side of the membrane. Activation by Ca2+ required a concentration of at least 10 microM and was maximal at 0.1 mM. Ba2+ did not substitute for Ca2+ in inducing channel activity nor did it inhibit activation by Ca2+. Increasing the concentration of intracellular Mg2+ stabilized the open state of NAD(+)-activated channels. 6. The non-selective cation channel reported here differs in its gating and modulatory characteristics from non-selective cation channels described in other tissues. This channel may play a role in the pathophysiological responses of beta-cells to oxidative stress.