Increase In Membrane Conductance Research Articles

Prolonged calcium influx after termination of light-induced calcium release in invertebrate photoreceptors

In microvillar photoreceptors, light stimulates the phospholipase C cascade and triggers an elevation of cytosolic Ca2+ that is essential for the regulation of both visual excitation and sensory adaptation. In some organisms, influx through light-activated ion channels contributes to the Ca2+ increase. In contrast, in other species, such as Lima, Ca2+ is initially only released from an intracellular pool, as the light-sensitive conductance is negligibly permeable to calcium ions. As a consequence, coping with sustained stimulation poses a challenge, requiring an alternative pathway for further calcium mobilization. We observed that after bright or prolonged illumination, the receptor potential of Lima photoreceptors is followed by the gradual development of an after-depolarization that decays in 1–4 minutes. Under voltage clamp, a graded, slow inward current (Islow) can be reproducibly elicited by flashes that saturate the photocurrent, and can reach a peak amplitude in excess of 200 pA. Islow obtains after replacing extracellular Na+ with Li+, guanidinium, or N-methyl-d-glucamine, indicating that it does not reflect the activation of an electrogenic Na/Ca exchange mechanism. An increase in membrane conductance accompanies the slow current. Islow is impervious to anion replacements and can be measured with extracellular Ca2+ as the sole permeant species; Ba can substitute for Ca2+ but Mg2+ cannot. A persistent Ca2+ elevation parallels Islow, when no further internal release takes place. Thus, this slow current could contribute to sustained Ca2+ mobilization and the concomitant regulation of the phototransduction machinery. Although reminiscent of the classical store depletion–operated calcium influx described in other cells, Islow appears to diverge in some significant aspects, such as its large size and insensitivity to SKF96365 and lanthanum; therefore, it may reflect an alternative mechanism for prolonged increase of cytosolic calcium in photoreceptors.

‘Leaky’ neurons in the epileptic hippocampus: should we get excited?

‘Leaky’ neurons in the epileptic hippocampus: should we get excited?

Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

Light-transduction in melanopsin-expressing photoreceptors of Amphioxus

Spatial vision in different organisms is mediated by 2 classes of photoreceptors: microvillar and ciliary. Recently, additional photosensitive cells implicated in nonvisual light-dependent functions have been identified in the mammalian retina. A previously undescribed photopigment, melanopsin, underlies these photoresponses, and it has been proposed that its transduction mechanisms may be akin to the lipid-signaling scheme of invertebrate microvillar receptors, rather than the cyclic-nucleotide cascade of vertebrates. Melanopsin has an ancient origin in deuterostomia, and expresses in 2 morphologically distinct classes of cells in the neural tube of Amphioxus, the most basal extant chordate: pigmented ocelli, and Joseph cells. However, to our knowledge, their physiology and alleged photosensitivity had never been investigated. We dissociated both types of cells, and conclusively demonstrated by patch-electrode recoding that they are primary photoreceptors; their receptor potential is depolarizing, accompanied by an increase in membrane conductance. The action spectrum peaks in the blue region, approximately 470 nm, similar to the absorption of melanopsin in vitro. The light-dependent conductance rectifies inwardly; Na and Ca are differentially implicated in the 2 cell types. Fluorescence Ca imaging reveals that photostimulation rapidly mobilizes calcium from internal stores. Intracellular 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate severely impairs the photoresponse, indicating that light-evoked Ca elevation is an important event in photoexcitation. These observations support the notion that the lineage of microvillar photoreceptors and its associated light-signaling pathway also evolved in the chordates. Thus, Joseph cells and pigmented ocelli of the Amphioxus may represent a link between ancestral rhabdomeric-like light sensors present in prebilaterians and the circadian photoreceptors of higher vertebrates.

Potassium channels

KCa= : calcium-activated potassium channels; Kir= : inward rectifier potassium channels; VGKC, KV= : voltage-gated K+ channels. Potassium (K+) channels have a critical role in cellular signaling in the nervous system. Over the past decade, 50 human genes encoding various K+ channels have been cloned, and there have been important advances in the elucidation of the subunit composition, stoichiometry, biophysical properties, and modulation of these channels. Genetic linkage analyses have led to the identification of many loci encoding K+ channel subunits that are associated with familial epilepsy syndromes as well as other disorders such as episodic ataxia/myokymia, long-QT syndromes, and congenital deafness.1 Antibodies against voltage-gated K+ channels (VGKC, KV) have been associated with autoimmune limbic encephalitis, neuromyotonia, and other neurologic disorders. Over the past several years, there has been increasing interest in developing drugs that modulate the activity of these channels.2 There have been comprehensive reviews on each of these topics.1–7 The focus of this article is on some K+ channels that have been linked with epilepsy. K+ channels are critical in regulation of neuronal excitability, axonal conduction, and neurotransmitter release.1,3–6,8,9 They also have a major role in control of the heart rate and excitability, epithelial electrolyte transport, cell volume regulation, and smooth muscle contraction. Potassium channels are membrane-spanning proteins that selectively allow flow of K+ ions across the cell membrane along their electrochemical gradient. An increase in membrane conductance to K+ ions causes neuronal hyperpolarization, decreases neuronal excitability, and reduces their firing frequency. There is a large number of K+ channels with distinct subcellular localization, biophysical properties, modulation, and pharmacologic profile.1,3,4–9 Potassium channels are polymers of α-subunits encoded by approximately 50 genes in humans. These subunits contain a homologous pore (P) segment selective for K+ ions and a gating mechanism that serves to switch between open and closed channel conformations. …

Control of output gain in CA1 pyramidal cells using somatic shunting inhibition

Event Abstract Back to Event Control of output gain in CA1 pyramidal cells using somatic shunting inhibition Introduction: Gain modulation refers to a change in the scaling between the input and output of a system. In neurobiology, it has been shown that gain control is a fundamental computation performed by numerous brain regions (1, 2). At the cellular level, gain control can manifests itself as a change in the slope of the spike frequency-current (F-I) relationship. Results from cortical pyramidal and cerebellar granule cells have shown that, unlike the subthreshold current-voltage (I-V) relationship, an increase in membrane conductance does not decrease the slope of the F-I relationship (3-7). Several studies, however, have shown that gain control can be implemented using membrane voltage fluctuations (3, 6). An assumption in investigating the relationship between gain and conductance has been that changes in membrane leak conductance do not alter the intrinsic firing dynamics of the neuron outside of the expected changes in membrane time constant and input resistance. Recent data, however, has shown that increased somatic conductance can profoundly alter intrinsic properties (8, 9). In the current study, we investigated the ability for different levels of constant leak conductance applied at the soma of CA1 pyramidal cells to modulate the gain of the F-I relationship without noise. Methods: Patch-clamp recordings from hippocampal CA1 pyramidal cells were implemented using standard in vitro slice electrophysiology. A leak conductance with a reversal of -65 mV was introduced at the soma using dynamic clamp. The effects of leak conductance on the spike output gain of CA1 pyramidal cells were quantified by measuring the steady-state F-I relationship in the presence of different levels of added leak conductance (5, 10 and 15 nS). Results and Conclusions: We find that increased conductance has a strong divisive effect on the steady-state F-I relationship of CA1 pyramidal cells. The reduction in gain with increased leak conductance is associated with a more depolarized operating voltage range and increase in spike threshold. The more depolarized operating voltage results in a progressive increase in the magnitude of spike frequency adaptation through increased Na+ conductance inactivation. It has been established that spike frequency adaptation has a divisive effect on the F-I relationship (10, 11). Hence, the increase in spike frequency adaptation with leak conductance provides a mechanism for gain control.

How do synapses measure milliseconds?

How do synapses measure milliseconds?

Dendritic excitability during increased synaptic activity in rat neocortical L5 pyramidal neurons

Recent years have seen increased study of dendritic integration, mostly in acute brain slices. However, due to the low background activity in brain slices the integration of synaptic input in slice preparations may not truly reflect conditions in vivo. To investigate dendritic integration, back-propagation of the action potential (AP) and initiation of the dendritic Ca(2+) spike we simultaneously recorded membrane potential at the soma and apical dendrite of layer 5 (L5) pyramidal neurons in quiescent and excited acute brain slices. After excitation of the brain slice the somatic input resistance decreased and the apparent passive space constant shortened. However, the back-propagating AP and dendritic Ca(2+) spike were robust during increased synaptic activity. The dendritic Ca(2+) spike was suppressed by the ionic composition of the bath solution required for slice excitation, suggesting that Ca(2+) spikes may be smaller in vivo than in the acute slice preparation. The results presented here suggest that, under the conditions of slice excitation examined in this study, the increased membrane conductance induced by activation of voltage-gated channels during back-propagation of the AP and dendritic Ca(2+) spike initiation is sufficiently larger than the membrane conductance at subthreshold potentials to allow these two regenerative dendritic events to remain robust over several levels of synaptic activity in the apical dendrite of L5 pyramidal neurons.

Random Stimulation of Spider Mechanosensory Neurons Reveals Long-Lasting Excitation by GABA and Muscimol

gamma-Aminobutyric acid type A (GABA(A)) receptor activation inhibits many primary afferent neurons by depolarization and increased membrane conductance. Deterministic (step and sinusoidal) functions are commonly used as stimuli to test such inhibition. We found that when the VS-3 mechanosensory neurons innervating the spider lyriform slit-sense organ were stimulated by randomly varying white-noise mechanical or electrical signals, their responses to GABA(A) receptor agonists were more complex than the inhibition observed during deterministic stimulation. Instead, there was rapid excitation, then brief inhibition, followed by long-lasting excitation. During the final excitatory phase, VS-3 neuron sensitivity to high-frequency signals increased selectively and their linear information capacity also increased. Using experimental and simulation approaches we found that the excitatory effect could also be achieved by depolarizing the neurons without GABA application and that excitation could override the inhibitory effect produced by increased membrane conductance (shunting). When the VS-3 neurons were exposed to bumetanide, an antagonist of the Cl(-) transporter NKCC1, the GABA-induced depolarization decreased without any change in firing rate, suggesting that the effects of GABA can be maintained for a long time without additional Cl(-) influx. Our results show that the VS-3 neuron's response to GABA depends profoundly on the type of signals the neuron is conveying while the transmitter binds to its receptors.

Composite Nanofiber Network Membranes for PEM Fuel Cells

A new class of proton-exchange membranes has been developed and characterized, where an interconnected network of proton conducting polymer nanofibers is embedded in an inert (non-conducting) polymer matrix. In the present study the nanofiber network membrane contained electrospun fibers from sulfonated poly(arylene ether sulfone) solutions (with/without sulfonated polyhedral oligomeric silsesquioxane, sPOSS). The membranes were characterized in terms of morphology, proton conductivity, and water uptake. In-plane proton conductivity was measured using a BekkTech cell that was placed in a controlled temperature/humidity chamber. The data show: (1) There was a modest increase in nanofiber > 50%.membrane conductivity when the IEC of the fibers was increased from 2.1 to 2.6 mmol/g (in the absence of sPOSS), (2) There was a substantial increase in membrane conductivity when sPOSS was added to the fibers, and (3) The proton conductivity of the sPOSS-loaded membranes was significantly higher than that for a commercial Nafion 212 sample at 30oC for a relative humidity

Pyramidal Neurons Switch From Integrators In Vitro to Resonators Under In Vivo-Like Conditions

During wakefulness, pyramidal neurons in the intact brain are bombarded by synaptic input that causes tonic depolarization, increased membrane conductance (i.e., shunting), and noisy fluctuations in membrane potential; by comparison, pyramidal neurons in acute slices typically experience little background input. Such differences in operating conditions can compromise extrapolation of in vitro data to explain neuronal operation in vivo. For instance, pyramidal neurons have been identified as integrators (i.e., class 1 neurons according to Hodgkin's classification of intrinsic excitability) based on in vitro experiments but that classification is inconsistent with the ability of hippocampal pyramidal neurons to oscillate/resonate at theta frequency since intrinsic oscillatory behavior is limited to class 2 neurons. Using long depolarizing stimuli and dynamic clamp to reproduce in vivo-like conditions in slice experiments, we show that CA1 hippocampal pyramidal cells switch from integrators to resonators, i.e., from class 1 to class 2 excitability. The switch is explained by increased outward current contributed by the M-type potassium current I(M), which shifts the balance of inward and outward currents active at perithreshold potentials and thereby converts the spike-initiating mechanism as predicted by dynamical analysis of our computational model. Perithreshold activation of I(M) is enhanced by the depolarizing shift in spike threshold caused by shunting and/or sodium channel inactivation secondary to tonic depolarization. Our conclusions were validated by multiple comparisons between simulation and experimental data. Thus even so-called "intrinsic" properties may differ qualitatively between in vitro and in vivo conditions.

An electrochemical approach tunes the electric property of benzoylferrocene-modified supported lipid membrane

A benzoylferrocene (BFc) supported 3-sn-phosphatidylcholine (PC) film electrode was prepared by casting the solution of BFc and PC in chloroform onto the surface of platinum (Pt). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) results showed that BFc, retained in the biological membrane, acted as a shuttle for electron transfer across the supported bilayer lipid membranes (s-BLMs). Doping of BFc increased membrane conductivity, while electrochemical oxidation of BFc greatly changed the membrane conductivity, the membrane impedance characterized by charge transfer resistance ( R ct) dramatically increased about 400 times (from 10.32 to 3919.67 kΩ). Interestingly, the electrochemical oxidized BFc buried in the membranes could be reduced by applying a low potential, and this led to recurrent of a conductive membrane. The conductivity of the s-BLMs could be controlled by the redox status of embedded BFc molecules. The approach provided a facile and novel way to electrochemically control the membrane conductance of s-BLMs by embedding BFc as a switchable redox mediator.

A plateau potential mediated by the activation of extrasynaptic NMDA receptors in rat hippocampal CA1 pyramidal neurons

Hippocampal pyramidal neurons express various extrasynaptic glutamate receptors. When glutamate spillover was facilitated by blocking glutamate uptake and fast synaptic transmission was blocked by antagonists of AMPA- and NMDA-type glutamate receptors and an ionotropic GABA receptor blocker, repetitive synaptic stimulation evoked a persistent membrane depolarization that consisted of an early Ca(2+)-independent component and a late Ca(2+)-dependent component. The early component, which we refer to as a plateau potential, had a half-width of 770 +/- 160 ms and a steady peak level of -9.54 +/- 3.50 mV. It was accompanied by an increase in membrane conductance, the I-V relationship of which showed a peak at -19.91 +/- 2.18 mV and reversal of the current at -4.32 +/- 2.13 mV, and was suppressed by high concentration of an NMDA receptor (NMDAR) antagonist d-APV, or an NMDAR glycine-binding site antagonist 5,7-dCK. After blocking synaptically located NMDARs using MK801, the potential was still evoked synaptically when spillover was facilitated. A sustained depolarization was evoked by iontophoretic application of glutamate in the presence or absence of a glutamate uptake blocker. This potential was not affected by Na(+) or Ca(2+) channel blockers, but was suppressed by 5,7-dCK, leaving an unspecified depolarizing potential. Iontophoresis of NMDA evoked a sustained depolarization that was blocked by a high concentration of d-APV or 5,7-dCK. The I-V relationship of the current during this potential was similar to that obtained during the synaptically induced plateau potentials. These results show that CA1 pyramidal neurons generate plateau potentials mediated most likely by activation of extrasynaptic NMDARs.

Activation of GIRK channels in substantia gelatinosa neurones of the adult rat spinal cord: a possible involvement of somatostatin

Recent studies have suggested that spinal G-protein-coupled, inwardly rectifying K(+) (GIRK) channels play an important role in thermal nociception and the analgesic actions of morphine and other agents. In this study, we show that spinal GIRK channels are activated by an endogenous neurotransmitter using whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in adult rat spinal cord slices. Although repetitive stimuli applied to the dorsal root did not induce any slow responses, ones focally applied to the spinal dorsal horn produced slow inhibitory postsynaptic currents (IPSCs) at a holding potential of -50 mV in about 30% of the SG neurones recorded. The amplitude and duration of slow IPSCs increased with the number of stimuli and decreased with removal of Ca(2+) from the external Krebs solution. Slow IPSCs were associated with an increase in membrane conductance; their polarity was reversed at a potential close to the equilibrium potential for K(+), calculated from the Nernst equation. Slow IPSCs were blocked by addition of GDP-beta-S into the patch-pipette solution, reduced in amplitude in the presence of Ba(2+), and significantly suppressed in the presence of an antagonist of GIRK channels, tertiapin-Q. Somatostatin produced an outward current in a subpopulation of SG neurones and the slow IPSC was occluded during the somatostatin-induced outward current. Moreover, slow IPSCs were significantly inhibited by the somatostatin receptor antagonist cyclo-somatostatin. These results suggest that endogenously released somatostatin may induce slow IPSCs through the activation of GIRK channels in SG neurones; this slow synaptic transmission might play an important role in spinal antinociception.

Flufenamic Acid Affects Multiple Currents and Causes Intracellular Ca2+Release inAplysiaBag Cell Neurons

Flufenamic acid (FFA) is a nonsteroidal antiinflammatory agent, commonly used to block nonselective cation channels. We previously reported that FFA potentiated, rather than inhibited, a cation current in Aplysia bag cell neurons. Prompted by this paradoxical result, the present study examined the effects of FFA on membrane currents and intracellular Ca2+ in cultured bag cell neurons. Under whole cell voltage clamp, FFA evoked either outward (I out) or inward (I in) currents. I out had a rapid onset, was inhibited by the K+ channel blocker, tetraethylammonium, and was associated with both an increase in membrane conductance and a negative shift in the whole cell current reversal potential. I in developed more slowly, was inhibited by the cation channel blocker, Gd3+, and was concomitant with both an increased conductance and positive shift in reversal potential. FFA also enhanced the use-dependent inactivation and caused a positive-shift in the activation curve of the voltage-dependent Ca2+ current. Furthermore, as measured by ratiometric imaging, FFA produced a rise in intracellular Ca2+ that persisted in the absence of extracellular Ca2+ and was reduced by depleting either the endoplasmic reticulum and/or mitochondrial stores. Ca2+ appeared to be involved in the activation of I in, as strong intracellular Ca2+ buffering effectively eliminated I in but did not alter I out. Finally, the effects of FFA were likely not due to block of cyclooxygenase given that the general cyclooxygenase inhibitor, indomethacin, failed to evoke either current. That FFA influences a number of neuronal properties needs to be taken into consideration when employing it as a cation channel antagonist.

Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release

Systemic osmoregulation is an integrated physiological process through which water intake and excretion are continuously balanced against salt intake and excretion to maintain the osmolality of the extracellular fluid near an optimal 'set-point' value. The behaviors (that is, thirst and sodium appetite) and renal responses (diuresis and natriuresis) that are modulated to mediate osmoregulatory homeostasis are mainly controlled by the nervous system. Appropriate regulation of these parameters depends in large part on specialized osmosensitive neurons, termed osmoreceptors, which convert changes in plasma osmolality into electrical signals that ultimately modulate effector functions to achieve homeostasis. Previous work has shown that mechanosensitive cation channels expressed in osmoreceptor neurons play a key role in the process of osmosensory transduction. Although the molecular identity of these channels remains unknown, a growing body of evidence, reviewed here, indicates that members of the transient receptor potential vanilloid family of ion channels may contribute to osmosensory transduction and to homeostatic responses implicated in the control of water balance.

TRPV1 Gene Required for Thermosensory Transduction and Anticipatory Secretion from Vasopressin Neurons during Hyperthermia

Increases in core body temperature promote thermoregulatory cooling by stimulating sweat production and preemptive renal water reabsorption through the release of vasopressin (VP, antidiuretic hormone). The mechanism by which the hypothalamus orchestrates this anticipatory VP release during hyperthermia is unknown but has been linked to a central thermosensory mechanism. Here, we report that thermal stimuli spanning core body temperatures activate a calcium-permeable, ruthenium red- and SB366791-sensitive nonselective cation conductance in hypothalamic VP neurons. This response is associated with a depolarizing receptor potential and an increase in action potential firing rate, indicating that these neurons are intrinsically thermosensitive. The thermosensitivity of VP neurons isolated from trpv1 knockout (Trpv1(-/-)) mice was significantly lower than that of wild-type counterparts. Moreover, Trpv1(-/-) mice showed an impaired VP response to hyperthermia in vivo. Channels encoded by the trpv1 gene thus confer thermosensitivity in central VP neurons and contribute to the thermal control of VP release in vivo.

Epithelial Barrier Modulation by a Channel Forming Peptide

NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (Isc) was <3 microA cm(-2) for T-84, IPEC-J2 and PVD9902 cell monolayers and <8 microA cm(-2) for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in Isc to >15 microA cm(-2), indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm(-2): T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.

Effects of the muscarinic agonist, 5-methylfurmethiodide, on contraction and electrophysiology of Ascaris suum muscle

Contraction and electrophysiological effects of 5-methylfurmethiodide (MFI), a selective muscarinic agonist in mammals, were tested on Ascaris suum muscle strips. In a contraction assay, MFI produced weak contraction and was less potent than levamisole and acetylcholine. Atropine (3μM) a non-selective muscarinic antagonist in mammalian preparations, did not affect contractions produced by MFI. Mecamylamine (3μM) a nicotinic antagonist in A. suum preparations, blocked the MFI contractions indicating that MFI had weak nicotinic agonist actions. In two-micropipette current-clamp experiments MFI, at concentrations greater than 10μM, produced concentration-dependent depolarizations and small increases in membrane conductance. The depolarizing effects were not abolished by perfusing the preparation in a calcium-free Ascaris Ringer solution to block synaptic transmission, suggesting that MFI effects were mediated by receptors on the muscle and were calcium-independent. A high concentration of mecamylamine, 30μM, only reduced the depolarizing responses by 42%, indicating that MFI also had effects on non-nicotinic receptors. Three non-nicotinic effects in the presence of 30μM mecamylamine were identified using voltage-clamp techniques: (i) MFI produced opening of mecamylamine-resistant non-selective-cation channel currents; (ii) MFI inhibited opening of voltage-activated potassium currents; and (iii) MFI increased the threshold of voltage-activated calcium currents. We suggest that a drug that is more selective for voltage-activated potassium currents, without effects on other channels like MFI, may be exploited pharmacologically as a novel anthelmintic or as an agent to potentiate the action of levamisole. In a larval migration assay we demonstrated that 4-aminopyridine (4-AP: a potassium channel blocker) potentiated the effects of levamisole but MFI did not.

Subunits α, β and γ of the epithelial Na+ channel (ENaC) are functionally related to the hypertonicity-induced cation channel (HICC) in rat hepatocytes

Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits α, β, and γ of the epithelial Na+ channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 → 400 mosM) increased membrane conductance from 75.4 ± 9.4 to 91.1 ± 11.2 pS (p < 0.001). The effect was completely blocked by 100 μM amiloride and reduced to 46, 30, and 45% of the control value by anti-α-, anti-β-, and anti-γ-rENaC siRNA, respectively. Scanning acoustic microscopy revealed an initial shrinkage of cells from 6.98 ± 0.45 to 6.03 ± 0.43 pl within 2 min. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 ± 0.05 pl (p < 0.001). With anti-α-, anti-β-, and anti-γ-rENaC siRNA, the volume response was reduced to 31, 31, and 36% of the reference level, respectively. It is concluded that all three subunits of the ENaC are functionally related to RVI and HICC activation in rat hepatocytes.