scholarly journals Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.

1995 ◽  
Vol 105 (2) ◽  
pp. 267-287 ◽  
Author(s):  
J McHugh ◽  
W M Mok ◽  
G K Wang ◽  
G Strichartz
Keyword(s):  
Local Anesthetic ◽  
Uv Light ◽  
Sodium Current ◽  
Covalent Bond ◽  
Cell Voltage ◽  
Scorpion Venom ◽  
Gh3 Cells ◽  
Post Exposure ◽  
Irreversible Inhibition ◽  
Uv Illumination

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Surface-attached Polymer Networks

2000 ◽  
Vol 629 ◽  
Author(s):  
Oswald Prucker ◽  
Kristin Müller ◽  
Jürgen Rühe
Keyword(s):  
Uv Light ◽  
Polymer Networks ◽  
Covalent Bond ◽  
Thermal Polymerization ◽  
Synthetic Route ◽  
Present Evidence ◽  
The Third ◽  
Uv Illumination ◽  
Glass Surfaces

ABSTRACTIn this paper we present three novel routes for the preparation of surface-attached polymer networks. In one system the network is formed in situ at the surface by thermal polymerization in solution and from the surface. Another synthetic route starts with a surface that carries photoreactive groups. Onto this surface a polymer is deposited that also carries photoreactive groups and both the crosslinks of the network and the covalent bond to the surface are formed by UV illumination. The third approach start with the in situ formation of surface-attached copolymers that again carry photoreactive groups that are subsequently linked together by UV light. We present evidence for the successful synthesis of these networks and their superior adhesion on glass surfaces.

Sapecin B, a novel fly toxin, blocks macroscopic K+ currents in the GH3 rat pituitary cell line

1997 ◽  
Vol 273 (1) ◽  
pp. C289-C296 ◽  
Author(s):  
N. Suzuki ◽  
M. Hirono ◽  
K. Kawahara ◽  
T. Yoshioka
Keyword(s):  
Inhibitory Concentration ◽  
Cell Voltage ◽  
Scorpion Venom ◽  
Bk Channels ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Dependent Manner ◽  
20 Nm ◽  
Dose Dependent ◽  
K Currents

Sapecin B is structurally homologous to charybdotoxin (CTX), which is found in scorpion venom. This study investigated the effects of sapecin B on the Ca(2+)-activated K+ currents [IK(Ca)] and the rapidly inactivating K+ currents in clonal rat GH3 pituitary cells with whole cell voltage-clamp methods. Sapecin B (20 nM) reversibly blocked the CTX-sensitive Ix(Ca) (the BK current) in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 0.9 nM, comparable to that of 0.08-0.4 nM for CTX. The Ca2+ currents in GH3 cells, however, were not affected by sapecin B (40 nM), indicating that the blockade of IK(Ca) by sapecin B is not a secondary effect of Ca2+ current inhibition. The effect of sapecin B on IK(Ca) resembled that of CTX, as expected from the structural similarities shared by CTX and sapecin B. We also found that sapecin B largely inhibited the 4-aminopyridine-sensitive, rapidly inactivating K+ currents in a dose-dependent manner, with a half-maximal inhibitory concentration of approximately 40 nM, whereas CTX had little effect on this current in GH3 cells. Sapecin B may thus provide a useful tool, complementary to CTX, for probing the functional role of molecular domains in the BK channels and the structural similarities common to the BK and the rapidly inactivating A-type K+ channels.

Local Anesthetic Properties of Prenylamine

2001 ◽  
Vol 95 (5) ◽  
pp. 1198-1204 ◽  
Author(s):  
Mustafa G. Mujtaba ◽  
Peter Gerner ◽  
Ging Kuo Wang
Keyword(s):  
Pain Management ◽  
Sciatic Nerve ◽  
Local Anesthetic ◽  
Cell Voltage ◽  
Gh3 Cells ◽  
Na Channels ◽  
Voltage Gated Sodium Channels ◽  
Nociceptive Responses

Background Local anesthetics that produce analgesia of long duration with minimal impairment of autonomic functions are highly desirable for pain management in the clinic. Prenylamine is a known calcium channel blocker, but its local anesthetic blocking effects on voltage-gated sodium channels have not been studied thus far. Methods The authors characterized the tonic and use-dependent prenylamine block of native Na(+) channels in cultured rat neuronal GH3 cells during whole cell voltage clamp conditions and the local anesthetic effect of prenylamine by neurologic evaluation of sensory and motor functions of sciatic nerve during neural block in rats. Results Prenylamine elicits both use-dependent block of Na(+) channels during repetitive pulses (3 microm prenylamine produced 50% block at 5 Hz) and tonic block for both resting and inactivated Na(+) channels. The 50% inhibitory concentration for prenylamine was 27.6 +/- 1.3 microm for resting channels and 0.75 +/- 0.02 microm for inactivated channels. Furthermore, in vivo data show that 10 mm prenylamine produced a complete sciatic nerve block of motor function, proprioceptive responses, and nociceptive responses that lasted approximately 27, 34, and 24 h, respectively. Rats injected with 15.4 mm bupivacaine, a known local anesthetic currently used for pain management, had a significantly shorter duration of blockade (< 2 h) compared with rats injected with prenylamine. Conclusions The data presented here demonstrate that prenylamine possesses local anesthetic properties in vitro and elicits prolonged local anesthesia in vivo.

scholarly journals Characterization of Direct Perturbations on Voltage-Gated Sodium Current by Esaxerenone, a Nonsteroidal Mineralocorticoid Receptor Blocker

Biomedicines ◽  
2021 ◽  
Vol 9 (5) ◽  
pp. 549
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Mineralocorticoid Receptor ◽  
Sodium Current ◽  
Ionic Currents ◽  
Antagonistic Effect ◽  
Gh3 Cells ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Ic50 Value ◽  
Low Threshold

Esaxerenone (ESAX; CS-3150, Minnebro®) is known to be a newly non-steroidal mineralocorticoid receptor (MR) antagonist. However, its modulatory actions on different types of ionic currents in electrically excitable cells remain largely unanswered. The present investigations were undertaken to explore the possible perturbations of ESAX on the transient, late and persistent components of voltage-gated Na+ current (INa) identified from pituitary GH3 or MMQ cells. GH3-cell exposure to ESAX depressed the transient and late components of INa with varying potencies. The IC50 value of ESAX required for its differential reduction in peak or late INa in GH3 cells was estimated to be 13.2 or 3.2 μM, respectively. The steady-state activation curve of peak INa remained unchanged during exposure to ESAX; however, recovery of peak INa block was prolonged in the presence 3 μM ESAX. In continued presence of aldosterone (10 μM), further addition of 3 μM ESAX remained effective at inhibiting INa. ESAX (3 μM) potently reversed Tef-induced augmentation of INa. By using isosceles-triangular ramp pulse with varying durations, the amplitude of persistent INa measured at high or low threshold was enhanced by the presence of tefluthrin (Tef), in combination with the appearance of the figure-of-eight hysteretic loop; moreover, hysteretic strength of the current was attenuated by subsequent addition of ESAX. Likewise, in MMQ lactotrophs, the addition of ESAX also effectively decreased the peak amplitude of INa along with the increased current inactivation rate. Taken together, the present results provide a noticeable yet unidentified finding disclosing that, apart from its antagonistic effect on MR receptor, ESAX may directly and concertedly modify the amplitude, gating properties and hysteresis of INa in electrically excitable cells.

Sodium Currents in Neurons From the Rostroventrolateral Medulla of the Rat

2003 ◽  
Vol 90 (3) ◽  
pp. 1635-1642 ◽  
Author(s):  
Ilya A. Rybak ◽  
Krzysztof Ptak ◽  
Natalia A. Shevtsova ◽  
Donald R. McCrimmon
Keyword(s):  
Sodium Channels ◽  
Sodium Current ◽  
Cell Voltage ◽  
Kinetic Properties ◽  
Persistent Sodium Current ◽  
Sodium Currents ◽  
Bötzinger Complex ◽  
Window Current ◽  
Bursting Activity

Rapidly inactivating and persistent sodium currents have been characterized in acutely dissociated neurons from the area of rostroventrolateral medulla that included the pre-Bötzinger Complex. As demonstrated in many studies in vitro, this area can generate endogenous rhythmic bursting activity. Experiments were performed on neonate and young rats (P1-15). Neurons were investigated using the whole cell voltage-clamp technique. Standard activation and inactivation protocols were used to characterize the steady-state and kinetic properties of the rapidly inactivating sodium current. Slow depolarizing ramp protocols were used to characterize the noninactivating sodium current. The “window” component of the rapidly inactivating sodium current was calculated using mathematical modeling. The persistent sodium current was revealed by subtraction of the window current from the total noninactivating sodium current. Our results provide evidence of the presence of persistent sodium currents in neurons of the rat rostroventrolateral medulla and determine voltage-gated characteristics of activation and inactivation of rapidly inactivating and persistent sodium channels in these neurons.

Insect Specific Neurotoxins from Scorpion Venom that Affect Sodium Current Inactivation

1994 ◽  
Vol 13 (1) ◽  
pp. 25-43 ◽  
Author(s):  
Eliahu Zlotkin ◽  
Michal Eitan ◽  
Marcel Pelhate ◽  
Nor Chejanovsky ◽  
Michael Gurevitz ◽  
...  
Keyword(s):  
Sodium Current ◽  
Scorpion Venom

Sodium current function in adult and aged canine atrial cells

2006 ◽  
Vol 291 (2) ◽  
pp. H756-H761 ◽  
Author(s):  
Shigeo Baba ◽  
Wen Dun ◽  
Masanori Hirose ◽  
Penelope A. Boyden
Keyword(s):  
Normal Sinus Rhythm ◽  
Sodium Current ◽  
Cell Voltage ◽  
Current Function ◽  
Structural Remodeling ◽  
Channel Protein ◽  
Normal Sinus ◽  
Atrial Cells ◽  
The Difference ◽  
In Cells

The incidence of atrial fibrillation increases with age, but it is unknown whether there are changes in the intrinsic function of Na+ currents in cells of the aged atria. Thus, we studied right (RA) and left (LA) atrial cells from two groups of dogs, adult and aged (>8 yr), to determine the change in Na+ currents with age. In this study all dogs were in normal sinus rhythm. Whole cell voltage clamp techniques were used to compare the Na+ currents in the two cell groups. Immunocytochemical studies were completed for the Na+ channel protein Nav1.5 to determine whether there was structural remodeling of this protein with age. In cells from aged animals, we found that Na+ currents are similar to those we measured in adult atria. However, Na+ current ( INa) density of the aged atria differed depending on the atrial chamber with LA cell currents being larger than RA cell currents. Thus with age, the difference in INa density between atrial chambers remains. INa kinetic differences between aged and adult cells included a significant acceleration into the inactivated state and an enhanced use-dependent decrease in peak current in aged RA cells. Finally, there is no structural remodeling of the cardiac Na+ channel protein Nav1.5 in the aged atrial cell. In conclusion, with age there is no change in INa density, but there are subtle kinetic differences contributing to slight enhancement of use dependence. There is no structural remodeling of the fast Na+ current protein with age.

scholarly journals Irreversible Inhibition of BoNT/A Protease: Unique Warhead Reactivity and Function Contingent upon a Bifunctional Approach

2021 ◽  
Author(s):  
Lewis Turner ◽  
Alexander Lund Nielsen ◽  
Lucy Lin ◽  
Sabine Pellett ◽  
Takashi Sugane ◽  
...  
Keyword(s):  
Enzyme Kinetics ◽  
Therapeutic Potential ◽  
Covalent Bond ◽  
Pharmacological Properties ◽  
Irreversible Inhibition ◽  
And Function ◽  
Chelating Compounds

We describe a comprehensive screening campaign of warheads, linked to a hydroxamate chelating anchor, for the modification of Cys165 within the BoNT/A protease. <div>Engaging thorough enzyme kinetics, we detail a remarkable proximity-driven covalent bond with an epoxide warhead, a weak electrophile; yet, one that possessed superior irreversible inhibition, and pharmacological properties, when compared to intrinsically higher reactive warheads. This directed, selective covalent bond was contingent upon the crucial hydroxamate-Zn<sup>2+ </sup>chelating interaction as exemplified by examining non-chelating compounds. </div><div>We discuss previous approaches using non-target selective cysteine-reactive warheads to modify the BoNT/A protease of which none present any therapeutic potential – our bifunctional strategy allows the use of intrinsically less reactive warheads to intercept the cysteine, which will allow for less off-target modifications of such inhibitors. Moreover, we also broach that this bifunctional approach is not a one-off strategy that we believe can be broadly translated to other metalloproteases that possess non-catalytic, yet, nucleophilic residues within the enzymes catalytic sphere. </div>

Optical and Electrical Characterizations of Nanoscale Robust 3C-SiC Membrane for UV Sensing Applications

2018 ◽  
Vol 775 ◽  
pp. 278-282
Author(s):  
A.R.M. Foisal ◽  
T. Dinh ◽  
A. Iacopi ◽  
L. Hold ◽  
E.W. Streed ◽  
...  
Keyword(s):  
Uv Light ◽  
Optical Characterization ◽  
Considerable Change ◽  
Electrical Contacts ◽  
Si Substrate ◽  
Uv Illumination ◽  
Sensing Applications ◽  
Transmission Measurements ◽  
Electrical Characterizations

This paper presents the fabrication and optical characterization of an ultrathin 3C-SiC membrane for UV light detection. SiC nanoscale film was grown on Si substrate and subsequently released to form a robust membrane with a high aspect ratio of about 5000. Transmission measurements were performed to determine the thickness of the film with a high accuracy of 98%. We also employed a simple and highly effective direct wirebonding technique to form electrical contacts to the SiC membrane. The considerable change in the photocurrent of the SiC membrane was observed under UV illumination, indicating the potential of using 3C-SiC membranes for UV detection.

Differences in voltage-dependent sodium currents exhibited by superficial and deep layer neurons of guinea pig entorhinal cortex

1994 ◽  
Vol 71 (5) ◽  
pp. 1986-1991 ◽  
Author(s):  
S. Fan ◽  
M. Stewart ◽  
R. K. Wong
Keyword(s):  
Guinea Pig ◽  
Entorhinal Cortex ◽  
Deep Layer ◽  
Sodium Current ◽  
Membrane Conductance ◽  
Cell Voltage ◽  
Superficial Layer ◽  
Peak Amplitude ◽  
Sodium Currents ◽  
Test Pulse

1. Sodium currents were studied using whole-cell voltage-clamp techniques in neurons acutely isolated from superficial (II/III) and deep (V/VI) layers of guinea pig entorhinal cortex. 2. Sodium currents were larger (peak amplitude) in superficial than in deep layer cells under the same conditions: -1939 +/- 780 (SD) pA (N = 6) versus -307 +/- 257 pA (N = 6). Specific membrane conductance was calculated to be 12.3 +/- 9.6 mS/cm2 for superficial layer cells and 1.4 +/- 0.9 mS/cm2 for deep layer cells. 3. Sodium currents could be activated in superficial layer cells from potentials as depolarized as -20 mV, whereas no significant currents could be activated in deep neurons from potentials more depolarized than about -50 mV. Using a protocol consisting of a 25-ms prepulse and a 20 ms test pulse, the inactivation curves for superficial layer cells were found to be shifted toward more depolarized potentials by an average of 15 mV (V50 = -59.8 +/- 3.8 mV compared with -75.7 +/- 12.0 mV for deep cells). This produced a region of overlap with the activation curves for superficial cells. 4. Over a range of about -50 to -20 mV in superficial layer cells, the region of overlap of the activation and inactivation curves, a sodium current could be activated, which did not fully inactivate during the test pulse (average peak amplitude: -89.5 +/- 48.7 pA; crossover voltage: -39.2 +/- 2.0 mV). Voltage steps to more depolarized potentials, outside the voltage “window”, permitted complete inactivation of the sodium current.(ABSTRACT TRUNCATED AT 250 WORDS)

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