Time Constant Of Inactivation Research Articles

Subunit‐specific inhibition of acid sensing ion channels by stomatin‐like protein 1

There are five mammalian stomatin-domain genes, all of which encode peripheral membrane proteins that can modulate ion channel function. Here we examined the ability of stomatin-like protein 1 (STOML1) to modulate the proton-sensitive members of the acid-sensing ion channel (ASIC) family. STOML1 profoundly inhibits ASIC1a, but has no effect on the splice variant ASIC1b. The inactivation time constant of ASIC3 is also accelerated by STOML1. We examined STOML1 null mutant mice with a β-galactosidase-neomycin cassette gene-trap reporter driven from the STOML1 gene locus, which indicated that STOML1 is expressed in at least 50% of dorsal root ganglion (DRG) neurones. Patch clamp recordings from mouse DRG neurones identified a trend for larger proton-gated currents in neurones lacking STOML1, which was due to a contribution of effects upon both transient and sustained currents, at different pH, a finding consistent with an endogenous inhibitory function for STOML1.

Contrasting alterations to synaptic and intrinsic properties in upper-cervical superficial dorsal horn neurons following acute neck muscle inflammation.

BackgroundAcute and chronic pain in axial structures, like the back and neck, are difficult to treat, and have incidence as high as 15%. Surprisingly, most preclinical work on pain mechanisms focuses on cutaneous structures in the limbs and animal models of axial pain are not widely available. Accordingly, we developed a mouse model of acute cervical muscle inflammation and assessed the functional properties of superficial dorsal horn (SDH) neurons.ResultsMale C57/Bl6 mice (P24-P40) were deeply anaesthetised (urethane 2.2 g/kg i.p) and the rectus capitis major muscle (RCM) injected with 40 μl of 2% carrageenan. Sham animals received vehicle injection and controls remained anaesthetised for 2 hrs. Mice in each group were sacrificed at 2 hrs for analysis. c-Fos staining was used to determine the location of activated neurons. c-Fos labelling in carrageenan-injected mice was concentrated within ipsilateral (87% and 63% of labelled neurons in C1 and C2 segments, respectively) and contralateral laminae I - II with some expression in lateral lamina V. c-Fos expression remained below detectable levels in control and sham animals. In additional experiments, whole cell recordings were obtained from visualised SDH neurons in transverse slices in the ipsilateral C1 and C2 spinal segments. Resting membrane potential and input resistance were not altered. Mean spontaneous EPSC amplitude was reduced by ~20% in neurons from carrageenan-injected mice versus control and sham animals (20.63 ± 1.05 vs. 24.64 ± 0.91 and 25.87 ± 1.32 pA, respectively). The amplitude (238 ± 33 vs. 494 ± 96 and 593 ± 167 pA) and inactivation time constant (12.9 ± 1.5 vs. 22.1 ± 3.6 and 15.3 ± 1.4 ms) of the rapid A type potassium current (IAr), the dominant subthreshold current in SDH neurons, were reduced in carrageenan-injected mice.ConclusionsExcitatory synaptic drive onto, and important intrinsic properties (i.e., IAr) within SDH neurons are reduced two hours after acute muscle inflammation. We propose this time point represents an important transition period between peripheral and central sensitisation with reduced excitatory drive providing an initial neuroprotective mechanism during the early stages of the progression towards central sensitisation.

Ion Channel Modulation in Spinal/Trigeminal Synaptic Plasticity

Ion Channel Modulation in Spinal/Trigeminal Synaptic Plasticity

Mutations in the Cavity Affect the Rate of Slow Inactivation in Shaker K+ Channels

It has been reported earlier that the activation and inactivation gates of Shaker channels are coupled, and this coupling might be mediated by a rotational motion of the S6 helix. We hypothese that either the electrostatics or the volume/hydrophobicity of the side chains pointing into the cavity can affect this coupling. Accordingly, we have introduced side-chains at position 470 in a T449A Shaker-IR background with different hydrophobicities according to the Wimley-White hydrophobicity scale (Ala, Cys, or Trp) and charges (Glu, Asp, Arg, Lys) and measured the inactivation kinetics. Mutant channels were transiently expressed in tsA_201 cells. All ionic current experiments were performed with excised inside-out patches. The introduction of an ionizable cysteine and the small but neutral alanine dramatically slowed entry into the inactivated state (the inactivation time constants for Shaker mutant T449A/I470C, T449A/I470A and T449A were 1.4 s, 250 ms and 50 ms, respectively). We could not detect any current on mutant T449A/I470X (X = Asp, Glu, Trp) but the transfection with a mixture of mutant and wild-type expression plasmids expressed currents. These heterotetramers had slower inactivation kinetics with respect to the T449A channels. Almost all of the channels had a midpoint voltage for activation in the range −50 mV to −20 mV. The only exception was the I470C mutant which had a large shift in voltage dependence (midpoint ∼5 mV). Mutant channels containing Arg or Lys (I470R, I470K) did not give functional expression either as homotetramers or as heterotetramers. Our results show that not the size but the physicochemical properties of the side chains (hydrophobicity and charge) at position 470 determine the inactivation kinetics of voltage-gated K+ channels, which may reflect interaction of the side chains with permeant ions.

Estragole blocks neuronal excitability by direct inhibition of Na+ channels

Estragole is a volatile terpenoid, which occurs naturally as a constituent of the essential oils of many plants. It has several pharmacological and biological activities. The objective of the present study was to investigate the mechanism of action of estragole on neuronal excitability. Intact and dissociated dorsal root ganglion neurons of rats were used to record action potential and Na+ currents with intracellular and patch-clamp techniques, respectively. Estragole blocked the generation of action potentials in cells with or without inflexions on their descendant (repolarization) phase (Ninf and N0 neurons, respectively) in a concentration-dependent manner. The resting potentials and input resistances of Ninf and N0 cells were not altered by estragole (2, 4, and 6 mM). Estragole also inhibited total Na+ current and tetrodotoxin-resistant Na+ current in a concentration-dependent manner (IC50 of 3.2 and 3.6 mM, respectively). Kinetic analysis of Na+ current in the presence of 4 mM estragole showed a statistically significant reduction of fast and slow inactivation time constants, indicating an acceleration of the inactivation process. These data demonstrate that estragole blocks neuronal excitability by direct inhibition of Na+ channel conductance activation. This action of estragole is likely to be relevant to the understanding of the mechanisms of several pharmacological effects of this substance.

Hypotonic Regulation of Mouse Epithelial Sodium Channel in Xenopus laevis Oocytes

The regulation of the epithelial Na⁺ channel (ENaC) during cell swelling is relevant in cellular processes in which cell volume changes occur, i.e., migration, proliferation and cell absorption. Its sensitivity to hypotonically induced swelling was investigated in the Xenopus oocyte expression system with the injection of the three subunits of mouse ENaC. We used voltage-clamp techniques to study the amiloride-sensitive Na⁺ currents (INa(amil)) and video microscopic methodologies to assess oocyte volume changes. Under conditions of mild swelling (25 % reduced hypotonicity) inward current amplitude decreased rapidly over 1.5 min. In contrast, there was no change in current amplitude of H₂O-injected oocytes to the osmotic insult. INa(amil) kinetics analysis revealed a decrease in the slower inactivation time constant during the hypotonic stimuli. Currents from ENaC-injected oocytes were not sensitive to external Cl⁻ reduction. Neither short- nor long-term cytochalasin D treatment affected the observed response. Oocytes expressing a DEG mutant β-ENaC subunit (β-S518K) with an open probability of 1 had reduced INa(amil) hypotonic response compared to oocytes injected with wild-type ENaC subunits. Finally, during the hypotonic response ENaC-injected oocytes did not show a cell volume difference compared with water-injected oocytes. On this basis we suggest that hypotonicity-dependent ENaC inhibition is principally mediated through an effect on open probability of channels in the membrane.

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K(+) channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1-4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12-17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19-21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.

Allocryptopine and benzyltetrahydropalmatine block hERG potassium channels expressed in HEK293 cells

Allocryptopine (ALL) is an alkaloid extracted from Corydalis decumbens (Thunb) Pers. Papaveraceae, whereas benzyltetrahydropalmatine (BTHP) is a derivative of tetrahydropalmatine extracted from Corydalis ambigua (Pall) Cham et Schlecht. The aim of this study was to investigate the effects of ALL and BTHP on the human ether-a-go-go related gene (hERG) current expressed in HEK293 cells. Cultured HEK293 cells were transiently transfected with hERG channel cDNA plasmid pcDNA3.1 using Lipofectamine. The whole-cell current IHERG was evoked and recorded using Axon MultiClamp 700B amplifier. The drugs were applied via supserfusion. Both ALL and BTHP reversibly suppressed the amplitude and density of IHERG in concentration- and voltage-dependent manners (the respective IC50 value was 49.65 and 22.38 μmol/L). BTHP (30 μmol/L) caused a significant negative shift of the steady-state inactivation curve of IHERG, while ALL (30 μmol/L) did not affect the steady-state inactivation of IHERG. Furthermore, BTHP, but not ALL, shortened the time constants of fast inactivation and slow time constants of deactivation of IHERG. But both the drugs markedly lengthened the time constants for recovery of IHERG from inactivation. Using action potential waveform pulses, it was found that both the drugs at 30 μmol/L significantly suppressed the current densities in the late phase of action potential, but did not significantly affect the current densities in the early phase of action potential. Both ALL and BTHP derived from Chinese herbs potently block hERG current.

Shab K+ channel slow inactivation

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h∞ curve. Regarding the mechanism of inactivation, we found that external K+ and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K+-dependent stability of the K+ conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K+, does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K+ conduction.

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca2+ current through both enhancement of Ca2+ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na+–H+ exchange (NHE), we examined the possible role of NHE in the regulation of ICa during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16dyn/cm2) fluid was applied onto single rat ventricular myocytes, and the ICa was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the ICa (IC50=0.97±0.12μM) without altering current–voltage relationships and inactivation time constants. In contrast, the ICa in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the ICa under FP was not altered by intracellular high Ca2+ buffering. Replacement of external Cl− with aspartate to inhibit the Cl−-dependent acid loader eliminated the inhibitory effect of HOE642 on ICa. These results suggest that NHE may attenuate FP-induced ICa suppression by preventing intracellular H+ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca2+-dependent inhibition of the ICa during FP exposure.

Kv3.4 Channel Expression, Modulation and Function in a Spinal Cord Injury Model of Neuropathic Pain

We previously showed that Kv3.4 channels underlie most of the high-voltage activated K+ current (IKHV) in dorsal root ganglion (DRG) nociceptors and elimination of Kv3.4 N-type inactivation by protein kinase C-dependent phosphorylation enhances the channel's ability to repolarize action potentials (APs) (Ritter et al. 2012 J Physiol 590:145-61). This function is important as reduction of Kv3.4 channel expression is found in diverse models of neuropathic pain. We hypothesize that phosphorylated Kv3.4 channels underlie an anti-nociceptive homeostatic mechanism in nociceptors that reduces downstream Ca++ signaling and down-regulation of Kv3.4 channel expression destroys this putative homeostatic mechanism, contributing to chronic pain. To test this hypothesis, we investigated a spinal cord injury (SCI) rat model of neuropathic pain in which a C5 hemi-contusion induces bilateral forepaw thermal hyperalgesia and mechanical allodynia 2 weeks post injury. In the contralateral SCI DRG, the peak IKHV is reduced by 55% and the time constant of inactivation (Taui) increases by 2-fold relative to laminectomy controls. Furthermore, while 85% of laminectomy nocicpetors show a fast inactivating Kv3.4 current, the SCI phenotypes fall into three categories: no current (25%), substantial sustained current (46%) or slow inactivation (29%, Taui >2 S.D. of controls). This suggests that Kv3.4 channels are phosphorylated and down-regulated in SCI. Confirming the impact of these changes on AP firing, AP-clamp experiments in SCI nociceptors show altered currents compared to controls. Interestingly, Kv3.4 mRNA splicing is altered in SCI DRG, which might alter the Kv3.4 channels' response to injury. We propose that Kv3.4 channels, their modulation by PKC and regulation of splicing are therapeutic targets in neuropathic pain.Supported by the Farber Family Foundation (MC), Sigma Xi GIAR (DR), Craig Nielson Foundation (AL), and Paralyzed Veterans of America (AL).

Temperature Enhances Activation and Inactivation Kinetics of Potassium Currents in Inner Hair Cells Isolated from Guinea-Pig Cochlea

ObjectivesUntil recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs.MethodsIHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25℃) and physiological temperatures (35℃-37℃).ResultsIHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36℃ than at room temperature. Half-time for activation was shorter at 36℃ than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q10 for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36℃ than at room temperature between the membrane potentials of -20 and +60 mV. Q10 for the inactivation time constant was 3.19.ConclusionThe results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.

Modeling the Structure and Function of L-Type Calcium Channel (Cav1.2) to Understand its Effect in Cardiac Propagation

The gating dynamics of the voltage gated L-type calcium channel (VGLCC) play an important role in intracellular calcium dynamics and the shape of the cardiac action potential. In this work we focus on modeling the structure of the VGLCC at the atomistic level to elucidate its structure-function relationship. using the crystal structure of the wild type voltage gated sodium channel (NavAb) i as the structural template, we propose a model of the Cav1.2 calcium channel. By using Molecular Dynamics simulations of the calcium channel embedded in a model membrane, we have explored the microscopic structure-function relationship of this channel protein. In parallel, we investigated the function of the channel in ventricular propagation dynamics. Our functional goal is to understand the effect of Cav1.2 gating in the ventricular tachycardia to fibrillation transition. With this in mind, we simulated the effect of changing the gating dynamics of the Cav1.2 on two dimensional spiral wave simulations run on a 640x640 grid wherein each node was represented by a Luo-Rudy model of the cardiac cellii. Specifically, we investigated and demonstrated the hypothesis that affecting the time constant of VGLCC inactivation destabilizes spiral wave behavior which is important in the clinical transition from normal rhythm to reentry tachycardia (one spiral) which often degenerates into ventricular fibrillation (multiple spirals). We will present our channel model and its validation to derive hypotheses of how our structure relates to the functional findings.Payandeh J et.al. Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature. 2012 May 20;486(7401):135-9.[1] Luo CH, Rudy Y. A dynamic model of the cardiac ventricular action potential. I. Simulations of ionic currents and concentration changes. Circ Res. 1994 Jun;74(6):1071-96.

Ginkgo biloba Extract and Ginkgolide Antiarrhythmic Potential by Targeting hERG and ICa-L Channel

We investigated the effects of Ginkgo biloba extract (GBE) and ginkgolide (GLD) on human ether-a-go-go-related gene (hERG)-encoded K+ channels and its underlying mechanisms in the hERG-HEK293 cell line by determining GBE- and GLD-induced changes in action potential duration (APD), L-type calcium currents (ICa-L), and the intracellular calcium concentration ([Ca2+]i) in guinea-pig ventricular myocytes. hERG currents, APD and ICa-L were recorded using the whole-cell patch clamp technique, the [Ca2+]i was examined by an immunofluorescence experiment. In the present study, we found that a low concentration of GBE (0.005 mg/ml) increased hERG currents, but the high concentration of GBE (from 0.05 to 0.25 mg/ml) reduced hERG currents. GLD reduced hERG currents in a concentration-dependent manner (from 0.005 to 0.25 mg/ml). Both GBE and GLD altered kinetics of the hERG channel. GBE accelerated the activation of hERG channels without changing the inactivation curve, but reduced the time constant of inactivation; GLD did not shift the activation or the inactivation curve, but only reduced the time constant of inactivation. Both GBE and GLD shortened the APD, inhibited the ICa-L currents, and decreased the [Ca2+]i in isolated guinea-pig ventricular myocytes. The results indicate that GBE and GLD can prevent ischemic arrhythmias and have an antiarrhythmic effect potential via inhibition of IKr and ICa-L currents.

Taxifolin Glycoside Blocks Humanether-a-go-goRelated Gene K+Channels

Taxifolin glycoside is a new drug candidate for the treatment of atopic dermatitis (AD). Many drugs cause side effects such as long QT syndrome by blocking the human ether-a-go-go related gene (hERG) K+ channels. To determine whether taxifolin glycoside would block hERG K+ channels, we recorded hERG K+ currents using a whole-cell patch clamp technique. We found that taxifolin glycoside directly blocked hERG K+ current in a concentration-dependent manner (EC50=9.6±0.7 µM). The activation curve of hERG K+ channels was negatively shifted by taxifolin glycoside. In addition, taxifolin glycoside accelerated the activation time constant and reduced the onset of the inactivation time constant. These results suggest that taxifolin glycoside blocks hERG K+ channels that function by facilitating activation and inactivation process.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

Inactivating ion channels augment robustness of subthreshold intrinsic response dynamics to parametric variability in hippocampal model neurons

Voltage-gated ion channels play a critical role in regulating neuronal intrinsic response dynamics (IRD). Here, we computationally analysed the roles of the two inactivating subthreshold conductances (A and T), individually and in various combinations with the non-inactivating h conductance, in regulating several physiological IRD measurements in the theta frequency range. We found that the independent presence of a T conductance, unlike that of an h conductance, was unable to sustain an inductive phase lead in the theta frequency range, despite its ability to mediate theta frequency resonance. The A conductance, on the other hand, when expressed independently, acted in a manner similar to a leak conductance with reference to most IRD measurements. Next, analysing the impact of pair-wise coexpression of these channels, we found that the coexpression of the h and T conductances augmented the range of parameters over which they sustained resonance and inductive phase lead. Additionally, coexpression of the A conductance with the h or the T conductance elicited changes in IRD measurements that were similar to those obtained with the expression of a leak conductance with a resonating conductance. Finally, to understand the global sensitivity of IRD measurements to all parameters associated with models expressing all three channels, we generated 100,000 neuronal models, each built with a unique set of parametric values. We categorized valid models among these by matching their IRD measurements with experimental counterparts, and found that functionally similar models could be achieved even when underlying parameters displayed tremendous variability and exhibited weak pair-wise correlations. Our results suggest that the three prominent subthreshold conductances contribute differently to intrinsic excitability and to phase coding. We postulate that the differential expression and activity-dependent plasticity of these conductances contribute to robustness of subthreshold IRD, whereby response homeostasis is achieved by recruiting several non-unique combinations of these channel parameters.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Chloride currents in cones modify feedback from horizontal cells to cones in goldfish retina

In neuronal systems, excitation and inhibition must be well balanced to ensure reliable information transfer. The cone/horizontal cell (HC) interaction in the retina is an example of this. Because natural scenes encompass an enormous intensity range both in temporal and spatial domains, the balance between excitation and inhibition in the outer retina needs to be adaptable. How this is achieved is unknown. Using electrophysiological techniques in the isolated retina of the goldfish, it was found that opening Ca(2+)-dependent Cl(-) channels in recorded cones reduced the size of feedback responses measured in both cones and HCs. Furthermore, we show that cones express Cl(-) channels that are gated by GABA released from HCs. Similar to activation of I(Cl(Ca)), opening of these GABA-gated Cl(-) channels reduced the size of light-induced feedback responses both in cones and HCs. Conversely, application of picrotoxin, a blocker of GABA(A) and GABA(C) receptors, had the opposite effect. In addition, reducing GABA release from HCs by blocking GABA transporters also led to an increase in the size of feedback. Because the independent manipulation of Ca(2+)-dependent Cl(-) currents in individual cones yielded results comparable to bath-applied GABA, it was concluded that activation of either Cl(-) current by itself is sufficient to reduce the size of HC feedback. However, additional effects of GABA on outer retinal processing cannot be excluded. These results can be accounted for by an ephaptic feedback model in which a cone Cl(-) current shunts the current flow in the synaptic cleft. The Ca(2+)-dependent Cl(-) current might be essential to set the initial balance between the feedforward and the feedback signals active in the cone HC synapse. It prevents that strong feedback from HCs to cones flood the cone with Ca(2)(+). Modulation of the feedback strength by GABA might play a role during light/dark adaptation, adjusting the amount of negative feedback to the signal to noise ratio of the cone output.

Single-molecule Analysis of Inhibitory Pausing States of V1-ATPase

V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V(1)-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V(1) in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F(1), suggesting that LP represents the ADP-inhibited state of V(1)-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V(1)-ATPase rotation modulates the off rate of ADP.