scholarly journals Use Dependent Attenuation of Rat HCN1-Mediated Ih in Intact HEK293 Cells

2016 ◽  
Vol 38 (6) ◽  
pp. 2079-2093 ◽  
Author(s):  
Lennart Barthel ◽  
Olivia Reetz ◽  
Ulf Strauss
Keyword(s):  
Mammalian Cells ◽  
Hek293 Cells ◽  
Hcn Channels ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Whole Cell ◽  
Human Embryonic Kidney Cells ◽  
Intracellular Content ◽  
Voltage Dependent ◽  
Activity Dependence

Background/Aims: Cationic currents (Ih) through the fast activating and relatively cAMP insensitive subtype of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, HCN1, are limited by cytosolic factors in mammalian cells. This cytosolic HCN1 break is boosted by changes in membrane voltage that are not characterized on a biophysical level, yet. Methods: We overexpressed rat (r)HCN1 in human embryonic kidney cells (HEK293) and recorded pharmacologically isolated Ih in cell-attached or whole-cell mode of the patch-clamp technique. Results: Recurring activation of rHCN1 reduced and slowed Ih in intact HEK293 cells (cell-attached mode). On the contrary, sustained disruption of the intracellular content (whole-cell mode) ceased activity dependence and partially enables voltage dependent hysteresis. The activity induced Ih attenuation in intact cells was independent of the main external cation, depended on the number of previous forced activations and was - at least in part - due to a shift in the voltage dependence of activation towards hyperpolarization as estimated by an adapted tail current analysis. Intracellular elevation of cAMP could not reverse the changes in Ih. Conclusion: Reduction of rHCN1 mediated Ih is use dependent and may involve the coupling of voltage sensor and pore.

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.

Functional Expression of Three P2X2 Receptor Splice Variants From Guinea Pig Cochlea

2000 ◽  
Vol 83 (3) ◽  
pp. 1502-1509 ◽  
Author(s):  
Chu Chen ◽  
Margarett S. Parker ◽  
Anthony P. Barnes ◽  
Prescott Deininger ◽  
Richard P. Bobbin
Keyword(s):  
Guinea Pig ◽  
Splice Variants ◽  
Reversal Potential ◽  
Functional Expression ◽  
Hek293 Cells ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Human Embryonic Kidney Cells ◽  
Guinea Pig Cochlea

ATP has been suggested to act as a neurotransmitter or a neuromodulator in the cochlea. The responses to ATP in different cell types of the cochlea vary in terms of the rate of desensitization and magnitude, suggesting that there may be different subtypes of P2X receptors distributed in the cochlea. Recently three ionotropic P2X2 receptor splice variants, P2X2–1, P2X2–2, and P2X2–3, were isolated and sequenced from a guinea pig cochlear cDNA library. To test the hypothesis that these different splice variants could be expressed as functional homomeric receptors, the three P2X2 receptor variants were individually and transiently expressed in human embryonic kidney cells (HEK293). The biophysical and pharmacological properties of these receptors were characterized using the whole cell patch-clamp technique. Extracellular application of ATP induced an inward current in HEK293 cells containing each of the three splice variants in a dose-dependent manner indicating the expression of homomeric receptors. Current-voltage ( I-V) relationships for the ATP-gated current show that the three subtypes of the P2X2 receptor had a similar reversal potential and an inward rectification index ( I 50 mV/ I −50 mV). However, the ATP-induced currents in cells expressing P2X2–1 and P2X2–2 variants were large and desensitized rapidly whereas the current in those cells expressing the P2X2–3 variant was much smaller and desensitized slower. The order of potency to ATP agonists was 2-MeSATP > ATP > α,β -MeATP for all three expressed splice variants. The ATP receptor antagonists suramin and PPADS reduced the effects of ATP on all three variants. Results demonstrate that three P2X2splice variants from guinea pig cochlea, P2X2–1, P2X2–2, and P2X2–3, can individually form nonselective cation receptor channels when these subunits are expressed in HEK293 cells. The distinct properties of these P2X2receptor splice variants may contribute to the differences in the response to ATP observed in native cochlear cells.

Modulation by endogenously released ATP and opioids of chromaffin cell calcium channels in mouse adrenal slices

2011 ◽  
Vol 300 (3) ◽  
pp. C610-C623 ◽  
Author(s):  
A. Hernández ◽  
P. Segura-Chama ◽  
N. Jiménez ◽  
A. G. García ◽  
J. M. Hernández-Guijo ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Chromaffin Cells ◽  
Opiate Receptors ◽  
Tonic Inhibition ◽  
Inactivation Kinetics ◽  
High Threshold ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Patch Configuration

Modulation of high-threshold voltage-dependent calcium channels by neurotransmitters has been the subject of numerous studies in cultures of neurons and chromaffin cells. However, no studies on such modulation exist in chromaffin cells in their natural environment, the intact adrenal medullary tissue. Here we performed such a study in voltage-clamped chromaffin cells of freshly prepared mouse adrenal slices under the whole cell configuration of the patch-clamp technique. The subcomponents of the whole cell inward Ca2+ current ( ICa) accounted for 49% for L-, 28% for N-, and 36% for P/Q-type channels. T-type Ca2+ channels or residual R-type Ca2+ currents were not seen. However, under the perforated-patch configuration, 20% of ICa accounted for a toxin-resistant R-type Ca2+ current. Exogenously applied ATP and methionine-enkephalin (Met-enk) inhibited ICa by 33%. Stop-flow and Ca2+ replacement by Ba2+, which favored the release of endogenous ATP and opioids, also inhibited ICa, with no changes in activation or inactivation kinetics. This inhibition was partially voltage independent and insensitive to prepulse facilitation. Furthermore, in about half of the cells, suramin and naloxone augmented ICa in the absence of exogenous application of ATP/Met-enk. No additional modulation of ICa was obtained after bath application of exogenous ATP and opioids to these already inhibited cells. Augmentation of ICa was also seen upon intracellular dialysis of guanosine 5′-[β-thio]diphosphate (GDPβS), indicating the existence in the intact slice of a tonic inhibition of ICa in resting conditions. These results suggest that in the intact adrenal tissue a tonic inhibition of ICa exists, mediated by purinergic and opiate receptors.

Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations

1994 ◽  
Vol 267 (4) ◽  
pp. C1152-C1159 ◽  
Author(s):  
S. Grinstein ◽  
R. Romanek ◽  
O. D. Rotstein
Keyword(s):  
Single Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Pipette Solution ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Patch Configuration ◽  
In Cells

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.

scholarly journals Multiple Forms of Endocytosis In Bovine Adrenal Chromaffin Cells

1997 ◽  
Vol 139 (4) ◽  
pp. 885-894 ◽  
Author(s):  
Corey Smith ◽  
Erwin Neher
Keyword(s):  
Chromaffin Cells ◽  
Initial Rate ◽  
Secretory Activity ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Whole Cell ◽  
Perforated Patch ◽  
Cell Configuration ◽  
Adrenal Chromaffin ◽  
Cell Capacitance

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ∼6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ∼248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.

N-Methyl-d-Aspartate–Induced Oscillations in Whole Cell Clamped Neurons From the Isolated Spinal Cord of Xenopus laevis Embryos

1999 ◽  
Vol 82 (2) ◽  
pp. 1069-1073 ◽  
Author(s):  
L. Prime ◽  
Y. Pichon ◽  
L. E. Moore
Keyword(s):  
Spinal Cord ◽  
Low Frequency ◽  
Oscillatory Activity ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Voltage Dependent ◽  
Whole Cell Recordings ◽  
External Calcium ◽  
Xenopus Laevis Embryos ◽  
Embryonic Neurons

The patch-clamp technique was used to measure the effect of N-methyl-d-aspartate (NMDA) on Xenopus embryonic neurons in an isolated, but intact spinal cord. Whole cell recordings were done at external calcium concentrations of 1 mM. NMDA alone (50–200 μM) or in association with 10 μM serotonin or glycine induced oscillatory activity in most presumed motoneurons, which were therefore considered part of rhythm generating networks. In the presence of TTX, one-half of these neurons maintained this activity. The oscillations fell into two main categories: voltage-dependent, low-frequency (0.3–0.5 Hz) and voltage-independent, high-frequency (3–8 Hz) oscillations. NMDA alone induced TTX-insensitive oscillations in one-third of the neurons; however, the percentage of neurons showing oscillations was greater in the presence of exogenous 5-hydroxytryptamine (5-HT) or glycine. Because these observations were made at embryonic stages where little or no serotonergic innervation exists, it is likely that NMDA-induced intrinsic oscillatory activity in Xenopus embryonic neurons does not require 5-HT.

scholarly journals Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels

2019 ◽  
Vol 20 (20) ◽  
pp. 4995 ◽  
Author(s):  
León-Aparicio ◽  
Salvador ◽  
Aparicio-Trejo ◽  
Briones-Herrera ◽  
Pedraza-Chaverri ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Rat Kidney ◽  
Atp Synthesis ◽  
Hek293 Cells ◽  
Immunogold Electron Microscopy ◽  
Hcn Channels ◽  
Inner Mitochondrial Membrane ◽  
Action Potential Firing ◽  
Human Embryonic Kidney Cells ◽  
Kidney Mitochondria

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1–4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.

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.

Voltage-Dependent Sodium and Calcium Currents in Acutely Isolated Adult Rat Trigeminal Root Ganglion Neurons

1999 ◽  
Vol 81 (3) ◽  
pp. 1123-1134 ◽  
Author(s):  
Hyung-Chan Kim ◽  
Man-Kyo Chung
Keyword(s):  
Calcium Currents ◽  
Inactivation Kinetics ◽  
Type I ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Adult Rat ◽  
Voltage Dependent ◽  
Trigeminal Root ◽  
Ganglion Neurons ◽  
Drug Free

Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons. Voltage-dependent sodium ( I Na) and calcium ( I Ca) currents in small (<30 μM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of I Na showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant I Na were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive I Na. Voltage-dependent I Ca was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of −80 mV evoked two distinct inward I Ca, low-voltage activated (LVA) and high-voltage activated (HVA) I Ca. LVA I Ca was first observed at −60 to −50 mV and reached a peak at about −30 mV. Amiloride (0.5 mM) suppressed ∼60% of the LVA I Ca, whereas ∼10% of HVA I Ca was inhibited by the same concentration of the amiloride. LVA I Ca was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA I Ca. The ω-conotoxin GVIA (ω-CgTx), an N-type I Ca blocker, suppressed ∼65% of the whole cell HVA I Ca at the concentration of 1 μM. The ω-CgTx-resistant HVA I Ca was sensitive to nifedipine (10 μM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by ∼25% of the drug-free control (∼70% of the ω-CgTx-resistant I Ca). The combination of 10 μM nifedipine and 1 μM ω-CgTx left ∼13% of the drug-free control I Ca unblocked. The DHP agonist S(−)-BayK8644 (5 μM) shifted the activation of the HVA I Ca to more negative potentials and increased its maximal amplitude. Additionally, S(−)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.

scholarly journals Src tyrosine kinase alters gating of hyperpolarization-activated HCN4 pacemaker channel through Tyr531

2008 ◽  
Vol 294 (1) ◽  
pp. C355-C362 ◽  
Author(s):  
Chen-Hong Li ◽  
Qi Zhang ◽  
Bunyen Teng ◽  
S. Jamal Mustafa ◽  
Jian-Ying Huang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Hcn Channels ◽  
Channel Kinetics ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Src Tyrosine Kinase ◽  
Whole Cell ◽  
Hek 293 ◽  
Whole Cell Patch Clamp ◽  
Pacemaker Channel

We recently discovered that the constitutively active Src tyrosine kinase can enhance hyperpolarization-activated, cyclic nucleotide-gated (HCN) 4 channel activity by binding to the channel protein. To investigate the mechanism of modulation by Src of HCN channels, we studied the effects of a selective inhibitor of Src tyrosine kinase, 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), on HCN4 and its mutant channels expressed in HEK 293 cells by using a whole cell patch-clamp technique. We found that PP2 can inhibit HCN4 currents by negatively shifting the voltage dependence of channel activation, decreasing the whole cell channel conductance, and slowing activation and deactivation kinetics. Screening putative tyrosine residues subject to phosphorylation yielded two candidates: Tyr531 and Tyr554. Substituting HCN4-Tyr531 with phenylalanine largely abolished the effects of PP2 on HCN4 channels. Replacing HCN4-Tyr554 with phenylalanine did not abolish the effects of PP2 on voltage-dependent activation but did eliminate PP2-induced slowing of channel kinetics. The inhibitory effects of HCN channels associated with reduced Src tyrosine activity is confirmed in HL-1 cardiomyocytes. Finally, we found that PP2 can decrease the heart rate in a mouse model. These results demonstrate that Src tyrosine kinase enhances HCN4 currents by shifting their activation to more positive potentials and increasing the whole cell channel conductance as well as speeding the channel kinetics. The tyrosine residue that mediates most of Src's actions on HCN4 channels is Tyr531.

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