Current Amplitude In Response Research Articles

Inhibitory synaptic transmission is impaired at higher extracellular Ca2+ concentrations in Scn1a+/\u2212 mouse model of Dravet syndrome

Dravet syndrome (DS) is an intractable form of childhood epilepsy that occurs in infancy. More than 80% of all patients have a heterozygous abnormality in the SCN1A gene, which encodes a subunit of Na+ channels in the brain. However, the detailed pathogenesis of DS remains unclear. This study investigated the synaptic pathogenesis of this disease in terms of excitatory/inhibitory balance using a mouse model of DS. We show that excitatory postsynaptic currents were similar between Scn1a knock-in neurons (Scn1a+/− neurons) and wild-type neurons, but inhibitory postsynaptic currents were significantly lower in Scn1a+/− neurons. Moreover, both the vesicular release probability and the number of inhibitory synapses were significantly lower in Scn1a+/− neurons compared with wild-type neurons. There was no proportional increase in inhibitory postsynaptic current amplitude in response to increased extracellular Ca2+ concentrations. Our study revealed that the number of inhibitory synapses is significantly reduced in Scn1a+/− neurons, while the sensitivity of inhibitory synapses to extracellular Ca2+ concentrations is markedly increased. These data suggest that Ca2+ tethering in inhibitory nerve terminals may be disturbed following the synaptic burst, likely leading to epileptic symptoms.

Facilitation of I Kr current by some hERG channel blockers suppresses early afterdepolarizations.

Drug-induced block of the cardiac rapid delayed rectifying potassium current (I Kr), carried by the human ether-a-go-go-related gene (hERG) channel, is the most common cause of acquired long QT syndrome. Indeed, some, but not all, drugs that block hERG channels cause fatal cardiac arrhythmias. However, there is no clear method to distinguish between drugs that cause deadly arrhythmias and those that are clinically safe. Here we propose a mechanism that could explain why certain clinically used hERG blockers are less proarrhythmic than others. We demonstrate that several drugs that block hERG channels, but have favorable cardiac safety profiles, also evoke another effect; they facilitate the hERG current amplitude in response to low-voltage depolarization. To investigate how hERG facilitation impacts cardiac safety, we develop computational models of I Kr block with and without this facilitation. We constrain the models using data from voltage clamp recordings of hERG block and facilitation by nifekalant, a safe class III antiarrhythmic agent. Human ventricular action potential simulations demonstrate the ability of nifekalant to suppress ectopic excitations, with or without facilitation. Without facilitation, excessive I Kr block evokes early afterdepolarizations, which cause lethal arrhythmias. When facilitation is introduced, early afterdepolarizations are prevented at the same degree of block. Facilitation appears to prevent early afterdepolarizations by increasing I Kr during the repolarization phase of action potentials. We empirically test this prediction in isolated rabbit ventricular myocytes and find that action potential prolongation with nifekalant is less likely to induce early afterdepolarization than action potential prolongation with dofetilide, a hERG channel blocker that does not induce facilitation. Our data suggest that hERG channel blockers that induce facilitation increase the repolarization reserve of cardiac myocytes, rendering them less likely to trigger lethal ventricular arrhythmias.

A single amino acid polymorphism in the Drosophila melanogaster Dα1 (ALS) subunit enhances neonicotinoid efficacy at Dα1-chicken β2 hybrid nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.

Polymorphisms are sometimes observed in native insect nicotinic acetylcholine receptor (nAChR) subunits, which are important insecticide targets, yet little is known of their impact on insecticide actions. Here we investigated the effects of a polymorphism involving the substitution of histidine108 by leucine in the Drosophila melanogaster Dα1 subunit on the agonist actions of the neurotransmitter acetylcholine (ACh) and two commercial neonicotinoid insecticides (imidacloprid and clothianidin). There was no significant impact of the H108L substitution on either the ACh EC50, the concentration leading to a half maximal ACh response, or the maximum current amplitude in response at 10 μM ACh, of the Dα1-chicken β2 nAChR expressed in Xenopus laevis oocytes. However, the response amplitudes to imidacloprid and clothianidin were significantly enhanced, indicating a role of His108 in the selective interactions of Dα1 with these neonicotinoids.

Cumulative Activation of Voltage-Dependent KVS-1 Potassium Channels

In this study, we reveal the existence of a novel use-dependent phenomenon in potassium channels, which we refer to as cumulative activation (CA). CA consists of an increase in current amplitude in response to repetitive depolarizing step pulses to the same potential. CA persists for up to 20 s and is similar to a phenomenon called "voltage-dependent facilitation" observed in some calcium channels. The KVS-1 K+ channel, which exhibits CA, is a rapidly activating and inactivating voltage-dependent potassium channel expressed in chemosensory and other neurons of Caenorhabditis elegans. It is unusual in being most closely related to the Shab (Kv2) family of potassium channels, which typically behave like delayed rectifier K+ channels in other species. The magnitude of CA depends on the frequency, voltage, and duration of the depolarizing step pulse. CA also radically changes the activation and inactivation kinetics of the channel, suggesting that the channel may undergo a physical modification in a use-dependent manner; thus, a model that closely simulates the behavior of the channel postulates the existence of two populations of channels, unmodified and modified. Use-dependent changes in the behavior of potassium channels, such as CA observed in KVS-1, could be involved in functional mechanisms of cellular plasticity such as synaptic depression that represent the cellular basis of learning and memory.

Subtype-specific actions of beta-amyloid peptides on recombinant human neuronal nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4) expressed in Xenopus laevis oocytes.

Two-electrode voltage-clamp electrophysiology has been used to study the actions of two amyloid peptides (Abeta(1-42), Abeta(1-40)) on alpha7, alpha4beta2 and alpha3beta4 recombinant human neuronal nicotinic acetylcholine receptors (nicotinic AChRs), heterologously expressed in Xenopus laevis oocytes. The application of Abeta(1-42) or Abeta(1-40) (1 pM-100 nM) for 5 s does not directly activate expressed human alpha7, alpha4beta2 or alpha3beta4 nicotinic AChRs.Abeta(1-42) and Abeta(1-40) are antagonists of alpha7 nicotinic AChRs. For example, 10 nM Abeta(1-42) and Abeta(1-40) both reduced the peak amplitude of currents recorded (3 mM ACh) to 48+/-5 and 45+/-10% (respectively) of control currents recorded in the absence of peptide. In both the cases the effect is sustained throughout a 30 min peptide application and is poorly reversible.Abeta(1-42) and Abeta(1-40) (10 nM) enhance currents recorded in response to ACh (3 mM) from oocytes expressing alpha4beta2 nicotinic AChRs by 195+/-40 and 195+/-41% respectively. This effect is transient, reaching a peak after 3 min and returning to control values after a 24 min application of 10 nM Abeta(1-42). We observe an enhancement of 157+/-22% of control ACh-evoked current amplitude in response to 100 nM Abeta(1-42) recorded from oocytes expressing alpha4beta2 nicotinic AChRs.Abeta(1-42) and Abeta(1-40) (10 nM) were without antagonist actions on the responses of alpha3beta4 nicotinic AChRs to ACh (1 nM-3 mM).

Saturation and self-inhibition of rat hippocampal GABA(A) receptors at high GABA concentrations.

Current responses to ultrafast gamma-aminobutyric acid (GABA) applications were recorded from excised patches in rat hippocampal neurons to study the gating properties of GABA(A) receptors at GABA concentrations close to saturating ones and higher. The amplitude of currents saturated at approximately 1 mm, while the onset rate of responses reached saturation at 4-6 mm GABA. At high GABA concentrations (> 10 mm), the amplitude of current responses was reduced in a dose-dependent manner with a half-blocking GABA concentration of approximately 50 mm. The peak reduction at high GABA doses was accompanied by a tendency to increase the steady-state to peak ratio. At concentrations higher than 30 mm, this effect took the form of a rebound current, i.e. during the prolonged GABA applications, the current firstly declined due to desensitization onset and then, instead of decreasing towards a steady-state value, clearly increased. Both the self-inhibition of GABA(A) receptors by high GABA doses and rebound were clearly voltage dependent, being larger at positive holding potentials. The fast desensitization component accelerated with depolarization at all saturating [GABA] tested. The rebound phenomenon indicates that the self-block of GABAA receptors is state dependent, and suggests that the sojourn in the desensitized conformation provides a 'rescue' from the block. We propose that high GABA concentrations inhibit the receptors by direct occlusion of the channel pore having no effect on the receptor gating.

Role of KCNQ1 in the cell swelling-induced enhancement of the slowly activating delayed rectifier K(+) current.

Cell swelling enhances a slowly activating delayed rectifier K(+) current (I(Ks)) in cardiac cells. This investigation was undertaken to determine which of the two structural units reconstituting the I(Ks) channel, KCNQ1 (KvLQT1) and KCNE1 (minK/IsK), plays a key role in the cell swelling-induced I(Ks) enhancement and to dissect a possible involvement of tyrosine phosphorylation therein. KCNQ1 was transiently expressed alone or together with KCNE1 in a heterologous mammalian cell line. Two distinct whole-cell membrane currents were separately observed during the exposure of transfected cells to various degrees of hyposmotic solutions. A hyposmotic challenge (0.7 times control osmolarity) resulted in about a twofold increase not only in the heteromeric KCNQ1/KCNE1, but also in the homomeric KCNQ1 channel currents. There was no significant difference in the incremental ratio of current amplitude in response to hyposmotic stress between the two KCNQ1-related currents, and the cells expressing the heteromeric channels swelled less than those with the homomeric channels or without the exogenous ones. The cell swelling-induced I(Ks) enhancement was not affected by a protein tyrosine kinase (PTK) inhibitor, by genistein (50 microM), or by an inhibitor of phosphotyrosine phosphatase (PTP), orthovanadate (500 microM), or a nonhydrolyzable ATP analogue, AMP-PNP (5 mM). Taken together, it is very likely that KCNQ1 might primarily participate in the I(Ks) enhancement by osmotic cell swelling. The obligatory dependence of the I(Ks) augmentation on PTK activity remained to be demonstrated, at least, in this expression system.

Functional differences between splice variants of the murine 5-HT 3A receptor: possible role for phosphorylation

The murine 5-HT 3A receptor subunit is expressed as either of two splice variants which are differentially regulated in vivo. The difference resides in a six-amino acid sequence within the cytoplasmic loop between transmembrane regions 3 and 4, which is present in the long form but not the short form. No physiological roles have yet been ascribed to the two splice variants. Whole cell patch clamp recording from transfected HEK 293 cells stably expressing either long or short form receptors showed very similar responses under control conditions. However, inclusion of 1 mM cAMP (activator of protein kinase A) in the patch pipette caused an initial increase in the desensitization rate of the long form, but a decrease in the short form. With the addition of 100 nM phorbol 12-myristate 13-acetate (PMA; activator of protein kinase C) to the pipette solution, responses elicited with 1 μM 5-HT revealed an increase in the current amplitude in the long but not the short form of the receptor. Over a longer time period, inclusion of PMA in the patch-pipette caused a faster run down of peak current amplitude in response to 30 μM 5-HT in the long form but did not affect the short form; there was no observed long-term effects of cAMP. We conclude that the long and short forms of the 5-HT 3 receptor are differentially modulated by agents that activate PKA and PKC. These different patterns of modulation could have markedly divergent consequences on receptor function.

Activation of a Ca2+-dependent K+ current in mouse fibroblasts by lysophosphatidic acid requires a pertussis toxin-sensitive G protein and Ras.

Lysophosphatidic acid (LPA) is a bioactive lipid that acts through G protein-coupled plasma membrane receptors and mediates a wide range of cellular responses. Here we report that LPA activates a K+ current in NIH3T3 mouse fibroblasts that leads to membrane hyperpolarization. The activation occurs with an EC50 value of 1.7 nM LPA. The K+ current is Ca2+-dependent, voltage-independent, and completely blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin with IC50 values of 1.7, 16, and 62 nM, respectively. The underlying K+ channels possess a single channel conductance of 33 pS in symmetrical K+ solution. Pretreatment of cells with pertussis toxin (PTX), Clostridium sordellii lethal toxin, or a farnesyl protein transferase inhibitor reduced the K+ current amplitude in response to LPA to about 25% of the control value. Incubation of cells with the protein tyrosine kinase inhibitor genistein or microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 reduced it by more than 50%. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase A activator 8-bromo-cAMP had no effect. These results indicate that the K+ channel activation by LPA is mediated by a signal transduction pathway involving a PTX-sensitive G protein, a protein tyrosine kinase, and Ras. LPA is already known to activate Cl- channels in various cell types, thereby leading to membrane depolarization. In conjunction with our results that demonstrate LPA-induced membrane hyperpolarization by activation of K+ channels, LPA appears to be significantly involved in the regulation of the cellular membrane potential.

Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels.

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration-dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations < 50 microM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 microM, and the blocking site is located 41 +/- 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 microM) and TPeA (20 microM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology. 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.

Beta-Adrenergic modulation of K+ current in human T lymphocytes

The whole-cell voltage-clamp technique was employed to study the beta-adrenergic modulation of voltage-gated K+ currents in CD8+ human peripheral blood lymphocytes. The beta-receptor agonist, isoproterenol, decreased the peak current amplitude and increased the rate of inactivation of the delayed rectifier K+ current. In addition, isoproterenol decreased the voltage dependence of steady-state inactivation and shifted the steady-state inactivation curve to the left. Isoproterenol, on the other hand, had no significant effect on the steady-state parameters of current activation. The isoproterenol-induced decrease in peak current amplitude was inhibited by the beta-blocker propranolol. Bath application of dibutyryl cAMP (1 mM) mimicked the effects of isoproterenol on both K+ current amplitude and time course of inactivation. Furthermore, the reduction in the peak current amplitude in response to isoproterenol was attenuated when PKI5-24 (2-5 microM), a synthetic peptide inhibitor of cAMP-dependent protein kinase, was present in the pipette solution. The increase in the rate of inactivation of the K+ currents in response to isoproterenol was mimicked by the internal application of GTP-gamma-S (300 microM) and by exposure of the cell to cholera toxin (1 microgram/ml), suggesting the involvement of a G protein. These results demonstrate that the voltage-dependent K+ conductance in T lymphocytes can be modulated by beta-adrenergic stimulation. The effects of beta-agonists, i.e., isoproterenol, appear to be receptor mediated and could involve cAMP-dependent protein kinase as well as G proteins. Since inhibition of the delayed rectifier K+ current has been found to decrease the proliferative response in T lymphocytes, the beta-adrenergic modulation of K+ current may well serve as a feedback control mechanism limiting the extent of cellular proliferation.

Cyclic nucleotide modulation of Na + and K + currents in the isolated node of Ranvier

The application of dibutyryl cyclic adenosine monophosphate (d-cAMP) to an isolated, voltage clamped node of Ranvier resulted in an increase in the peak inward and steady-state outward current amplitudes. Blocking K + channels with TEA abolished the effects of d-cAMP on Na + currents. Blocking Na + channels with TTX revealed an increase in K + current amplitude in response to d-cAMP. Application of a potent phosphodiesterase inhibitor, SQ20009, reproduced the effects of d-cAMP in the intact node, but with a latency in onset. These results suggest a physiological role for cAMP in the modulation of Na + and K + currents in myelinated peripheral nerve.