scholarly journals Segmental differences in firing properties and potassium currents in Drosophila larval motoneurons

2012 ◽  
Vol 107 (5) ◽  
pp. 1356-1365 ◽  
Author(s):  
Subhashini Srinivasan ◽  
Kimberley Lance ◽  
Richard B. Levine
Keyword(s):  
Patch Clamp ◽  
Abdominal Segment ◽  
Body Wall ◽  
Potassium Currents ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Body Wall Muscles ◽  
Motoneuron Activity ◽  
Firing Properties ◽  
Thoracic And Abdominal

Potassium currents play key roles in regulating motoneuron activity, including functional specializations that are important for locomotion. The thoracic and abdominal segments in the Drosophila larval ganglion have repeated arrays of motoneurons that innervate body-wall muscles used for peristaltic movements during crawling. Although abdominal motoneurons and their muscle targets have been studied in detail, owing, in part, to their involvement in locomotion, little is known about the cellular properties of motoneurons in thoracic segments. The goal of this study was to compare firing properties among thoracic motoneurons and the potassium currents that influence them. Whole-cell, patch-clamp recordings performed from motoneurons in two thoracic and one abdominal segment revealed both transient and sustained voltage-activated K+ currents, each with Ca++-sensitive and Ca++-insensitive [A-type, voltage-dependent transient K+ current (IAv)] components. Segmental differences in the expression of voltage-activated K+ currents were observed. In addition, we demonstrate that Shal contributes to IAv currents in the motoneurons of the first thoracic segment.

scholarly journals Slow inactivation of L-type calcium current distorts the measurement of L- and T-type calcium current in Purkinje myocytes.

1993 ◽  
Vol 102 (5) ◽  
pp. 859-869 ◽  
Author(s):  
N B Datyner ◽  
I S Cohen
Keyword(s):  
Patch Clamp ◽  
Calcium Current ◽  
Slow Inactivation ◽  
Total Calcium ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Method Of Measurement

We have examined slow inactivation of L-type calcium current in canine Purkinje myocytes with the whole cell patch clamp technique. Slow inactivation is voltage dependent. It is negligible at -50 mV but can inactivate more than half of available iCaL at -10 mV. There are two major consequences of this slow inactivation. First, standard protocols for the measurement of T-type current can dramatically overestimate its contribution to total calcium current, and second, the position and steepness of the inactivation versus voltage curve for iCaL will depend on the method of measurement. Given the widespread attempts to identify calcium current components and characterize them biophysically, an important first step should be to determine the extent of slow inactivation of calcium current in each preparation.

Whole-cell patch-clamp analysis of voltage-dependent calcium conductances in cultured embryonic rat hippocampal neurons

1989 ◽  
Vol 61 (3) ◽  
pp. 467-477 ◽  
Author(s):  
D. E. Meyers ◽  
J. L. Barker
Keyword(s):  
Patch Clamp ◽  
Hippocampal Neurons ◽  
Calcium Currents ◽  
Tetraacetic Acid ◽  
Whole Cell ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Cells Cultured ◽  
In Cells

1. Voltage-dependent calcium currents in embryonic (E18) hippocampal neurons cultured for 1-14 days were investigated using the whole-cell patch-clamp technique. 2. Calcium currents were isolated by removing K+ from both the internal and external solutions. In most recordings the external solution contained tetrodotoxin, tetraethylammonium ions, and low concentrations of Na+, whereas the internal solution contained the large cations and anions, N-methyl-D-glucamine and methanesulphonate, and an adenosine 5'-triphosphate (ATP) regenerating system (Forscher and Oxford, 1985) to retard “run-down” of Ca currents. 3. Under these conditions, the sustained inward current triggered during depolarizing steps was enhanced when extracellular [Ca2+] ([Ca2+]0) was raised from 2 to 10 mM and abolished when [Ca2+]0 was lowered to 0.1 mM or by addition of Co2+ ions. These results indicate that the inward current was carried primarily by Ca2+ ions and was designated ICa. This current may be comparable to the “high-voltage-activated” Ca current described in other preparations. 4. In cells cultured for 1-3 days, ICa was small or absent (less than 20 pA for cells 1 day in culture and less than 80 pA for cells 3 days in culture). Although ICa decayed considerably during depolarizing steps, there was little evidence of the transient calcium current (T current) that was recorded in approximately 40% of cells cultured longer than 6 days. Maximal (i.e., the largest) ICa increased from 20 to 80 pA in 1- to 3-day cells to 150–450 pA in cells cultured for longer than 6 days. 5. The decay of ICa elicited by depolarizations from holding potentials of -60 mV or more negative was usually greatest for the maximal ICa. Replacement of extracellular Ca2+ (4 mM) with Ba2+ (2 mM) resulted in a substantial decrease in the extent of decay of ICa and a shift of the I-V relation in the hyperpolarizing direction. 6. Qualitative data obtained from experiments in which different levels of internal Ca2+ buffering were employed demonstrated that, on average, the decay of ICa was reduced as the capacity and/or rate of buffering was increased. The mean decay of ICa in cells buffered with 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was 9 +/- 7 (SD) %, (n = 12) and 25 +/- 12%, (n = 12) for cells buffered with the same concentration of ethyleneglycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).(ABSTRACT TRUNCATED AT 400 WORDS)

Phorbol Ester-Induced Inhibition of Potassium Currents in Rat Sensory Neurons Requires Voltage-Dependent Entry of Calcium

2001 ◽  
Vol 85 (1) ◽  
pp. 362-373 ◽  
Author(s):  
Yi-Hong Zhang ◽  
J. L. Kenyon ◽  
G. D. Nicol
Keyword(s):  
Sensory Neurons ◽  
Phorbol Ester ◽  
Potassium Currents ◽  
Channel Blockers ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Rat Pups ◽  
Whole Cell Patch Clamp ◽  
Potassium Channel Blockers ◽  
Voltage Dependent

The whole cell patch-clamp technique was used to examine the effects of protein kinase C (PKC) activation (via the phorbol ester, phorbol 12,13 dibutyrate, PDBu) on the modulation of potassium currents ( I K) in cultured capsaicin-sensitive neurons isolated from dorsal root ganglia from embryonic rat pups and grown in culture. PDBu, in a concentration- and time-dependent manner, reduced I K measured at +60 mV by ∼30% if the holding potential ( V h) was −20 or −47 mV but had no effect if V h was −80 mV. The PDBu-induced inhibition of I K was blocked by pretreatment with the PKC inhibitor bisindolylmaleimide I and I K was unaffected by 4-α phorbol, indicating that the suppression of I Kwas mediated by PKC. The inhibition of I K by 100 nM PDBu at a V h of −50 mV was reversed over several minutes if V h was changed to −80 mV. In addition, intracellular perfusion with 5 mM bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid (BAPTA) or pretreatment with ω-conotoxin GVIA or Cd2+-Ringer, but not nifedipine, prevented the PDBu-induced suppression of I K at −50 mV, suggesting that a voltage-dependent influx of calcium through N-type calcium channels was necessary for the activation of PKC. The potassium channel blockers tetraethylammonium (TEA, 10 mM) and 4-aminopyridine (4-AP, 3 mM and 30 μM) reduced I K, but only TEA attenuated the ability of PDBu to further inhibit the current, suggesting that the I K modified by PDBu was sensitive to TEA. Interestingly, in the presence of 3 mM or 30 μM 4-AP, 100 nM PDBu inhibited I K when V h was −80 mV. Thus 4-AP promotes the capacity of PDBu to reduce I K at −80 mV. We find that activation of PKC inhibits I K in rat sensory neurons and that voltage-dependent calcium entry is necessary for the development and maintenance of this inhibition.

Electrophysiological characterization of a motilin agonist, GM611, on rabbit duodenal smooth muscle

1996 ◽  
Vol 271 (6) ◽  
pp. G1003-G1016
Author(s):  
K. Yamada ◽  
S. Chen ◽  
N. A. Abdullah ◽  
M. Tanaka ◽  
Y. Ito ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Patch Clamp ◽  
Monovalent Cation ◽  
Patch Clamp Technique ◽  
Spike Amplitude ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent ◽  
Potential Recording ◽  
Electrophysiological Characterization

Effects of motilin and a newly synthesized erythromycin derivative, GM611, on membrane potential and currents of rabbit duodenal smooth muscle have been investigated by intracellular potential recording and whole cell patch-clamp technique and compared with results from contractile experiments. Motilin and GM611 (0.01-100 nM) dose dependently produced slowly sustained depolarizations (half-maximal effective dose = 0.15 and 3.9 nM for motilin and GM611, respectively) but exhibited biphasic effects on spike activities superimposed on slow waves. With small depolarizations, the number of spike discharges increased, whereas larger depolarizations markedly reduced spike amplitude. Motilin-induced (or GM611-induced) depolarization appeared to be associated with the activation of monovalent cation-selective channels, and the reduction in the spike amplitude appeared mainly to be associated with inhibition of voltage-dependent Ca2+ channels. Furthermore, data from patch-clamp experiments suggested that Ca2+ release occurred from heparin-sensitive internal stores upon stimulation of motilin receptors by these agonists. Possible implications of these electrophysiological effects in motilin- or GM611-induced tonic and phasic contractions have been discussed.

scholarly journals Kvβ1.3 Reduces the Degree of Stereoselective Bupivacaine Block of Kv1.5 Channels

2007 ◽  
Vol 107 (4) ◽  
pp. 641-651 ◽  
Author(s):  
Cristina Arias ◽  
Miriam Guizy ◽  
Miren David ◽  
Stefanie Marzian ◽  
Teresa González ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Binding Site ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Time Dependent ◽  
Hek293 Cells ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

Background Kvbeta1.3 subunit modifies the gating and the pharmacology of Kv1.5 channels, decreasing their sensitivity to block induced by drugs, suggesting that Kvbeta1.3 competes with them for a binding site at Kv1.5 channels. Methods Currents generated by the activation of Kv1.5 and Kv1.5 + Kvbeta1.3 channels expressed in HEK293 cells and Xenopus oocytes were recorded by using whole cell patch clamp and voltage clamp techniques. Results Block of Kv1.5, but not that produced on Kv1.5 + Kvbeta1.3 channels, was voltage dependent. In both channels, bupivacaine block was time dependent. R(+)- and S(-)-bupivacaine blocked Kv1.5 with IC50 4.4 +/- 0.5 microM (n = 15) and 39.8 +/- 8.2 microM (n = 16; P < 0.05), respectively. These values increased fourfold for R(+)-bupivacaine (17.2 +/- 2.2 microM) and twofold for S(-)-bupivacaine (71.9 +/- 11.5 microM) in Kv1.5 + Kvbeta1.3 channels. Therefore, the degree of stereoselectivity (theta) decreased from 9 to 4 in the presence of Kvbeta1.3. The decrease in potency to block Kv1.5 + Kvbeta1.3 channels was the result of a less stable interaction between bupivacaine enantiomers and channels. Differences in stereoselectivity in each situation were due to a more favorable interaction between the channel and R(+)-bupivacaine. In the presence of Kvbeta1.3, stereoselectivity was abolished for V514A mutant channels (involved in bupivacaine binding but not in Kvbeta1.3 binding) but not for L510A (part of Kvbeta1.3 binding site). Conclusions The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.

Extracellular Ba2+ blocks the cardiac transient outward K+ current

2000 ◽  
Vol 278 (1) ◽  
pp. H295-H299 ◽  
Author(s):  
Hong Shi ◽  
Hui-Zhen Wang ◽  
Zhiguo Wang
Keyword(s):  
Patch Clamp ◽  
Inward Rectifier ◽  
Delayed Rectifier ◽  
Inactivation Kinetics ◽  
Atrial Myocytes ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Potent Inhibition ◽  
Voltage Dependent ◽  
K Current

Ba2+ is widely used as a tool in patch-clamp studies because of its ability to block a variety of K+channels and to pass Ca2+ channels. Its potential ability to block the cardiac transient outward K+ current ( I to) has not been clearly documented. We performed whole cell patch-clamp studies in canine ventricular and atrial myocytes. Extracellular application of Ba2+ produced potent inhibition of I to with an IC50 of ∼40 μM. The effects were voltage independent, and the inactivation kinetics were not altered by Ba2+. The potency of Ba2+ was ∼10 times higher than that of 4-aminopyridine (a selective I to blocker with an IC50 of 430 μM) under identical conditions. By comparison, Ba2+blockade of the inward rectifier K+ current was voltage dependent; the IC50 was ∼20 times lower (2.5 μM) than that for I to when determined at −100 mV and was comparable to I to as determined at −60 mV (IC50 = 26 μM). Ba2+ concentrations of ≤1 mM or higher failed to block ultrarapid delayed rectifier K+ current. Our data suggest that Ba2+ can be considered a potent blocker of I to.

Whole Cell Patch-Clamp Recordings of Rat Midbrain Dopaminergic Neurons Isolate a Sulphonylurea- and ATP-Sensitive Component of Potassium Currents Activated by Hypoxia

1998 ◽  
Vol 79 (3) ◽  
pp. 1239-1245 ◽  
Author(s):  
E. Guatteo ◽  
M. Federici ◽  
A. Siniscalchi ◽  
T. Knöpfel ◽  
N. B. Mercuri ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Dopaminergic Neurons ◽  
Outward Current ◽  
Potassium Currents ◽  
Whole Cell ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp ◽  
Midbrain Dopaminergic Neurons ◽  
Cytosolic Factors

Guatteo, E., M. Federici, A. Siniscalchi, T. Knöpfel, N. B. Mercuri, and G. Bernardi. Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia. J. Neurophysiol. 79: 1239–1245, 1998. The effects of brief (2–4 min) hypoxia on presumed dopaminergic “principal” neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode ( V h −60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 ± 15.2 (SE) pA ( n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 ± 10.6 pA ( n = 18). Although the hypoOUT reversed at −83.7 ± 3.8 mV ( n = 18), the posthypoOUT did not reverse. The K+ ATP-blocking sulphonylureas tolbutamide (100 μM) and glibenclamide (30 μM), significantly reduced the peak of the hypoOUT by 47.6 ± 7.7% ( n = 16) and 54.18 ± 7.5% ( n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 μM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10–50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings ( V h −60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 ± 14.5 pA that reversed polarity at −87.9 ± 5.1 mV ( n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 μM), glibenclamide (10–30 μM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 μM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.

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