Electrophysiological Characteristics of Rat Gustatory Cyclic Nucleotide–Gated Channel Expressed in Xenopus Oocytes

2001 ◽  
Vol 85 (6) ◽  
pp. 2335-2349 ◽  
Author(s):  
Han Mi Lee ◽  
Young Sun Park ◽  
Wonjae Kim ◽  
Chul-Seung Park
Keyword(s):  
Cyclic Nucleotide ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Divalent Cations ◽  
Epithelial Tissue ◽  
Dna Encoding ◽  
Open Probability ◽  
Voltage Dependent ◽  
Gated Channel ◽  
Channel Currents

The complementary DNA encoding gustatory cyclic nucleotide–gated ion channel (or gustCNG channel) cloned from rat tongue epithelial tissue was expressed in Xenopus oocytes, and its electrophysiological characteristics were investigated using tight-seal patch-clamp recordings of single and macroscopic channel currents. Both cGMP and cAMP directly activated gustCNG channels but with markedly different affinities. No desensitization or inactivation of gustCNG channel currents was observed even in the prolonged application of the cyclic nucleotides. Single-channel conductance of gustCNG channel was estimated as 28 pS in 130 mM of symmetric Na+. Single-channel current recordings revealed fast open-close transitions and longer lasting closure states. The distribution of both open and closed events could be well fitted with two exponential components and intracellular cGMP increased the open probability ( P o) of gustCNG channels mainly by increasing the slower opening rate. Under bi-ionic conditions, the selectivity order of gustCNG channel among divalent cations was determined as Na+ ∼ K+ > Rb+ > Li+ > Cs+ with the permeability ratio of 1:0.95:0.74:0.63:0.49. Magnesium ion blocked Na+ currents through gustCNG channels from both intracellular and extracellular sides in voltage-dependent manners. The inhibition constants ( K is) of intracellular Mg2+ were determined as 360 ± 40 μM at 70 mV and 8.2 ± 1.5 mM at −70 mV with zδ value of 1.04, while K is of extracellular Mg2+ were as 1.1 ± 0.3 mM at 70 mV and 20.0 ± 0.1 μM at −70 mV with zδ of 0.94. Although 100 μM l- cis-diltiazem blocked significant portions of outward Na+ currents through both bovine rod and rat olfactory CNG channels, the gustCNG channel currents were minimally affected by the same concentration of the drug.

Divalent cations block the cyclic nucleotide-gated channel of olfactory receptor neurons

1993 ◽  
Vol 69 (5) ◽  
pp. 1758-1768 ◽  
Author(s):  
F. Zufall ◽  
S. Firestein
Keyword(s):  
Cyclic Amp ◽  
Calcium Channels ◽  
Cyclic Nucleotide ◽  
Olfactory Receptor ◽  
Single Channel ◽  
Divalent Cations ◽  
Olfactory Receptor Neurons ◽  
Gated Channel ◽  
Receptor Neurons ◽  
Single Channel Currents

1. The effects of external divalent cations on odor-dependent, cyclic AMP-activated single-channel currents from olfactory receptor neurons of the tiger salamander (Ambystoma tigrinum) were studied in inside-out membrane patches taken from dendritic regions of freshly isolated sensory cells. 2. Channels were reversibly activated by 100 microM cyclic AMP. In the absence of divalent cations, the channel had a linear current-voltage relation giving a conductance of 45 pS. With increasing concentrations of either Ca2+ or Mg2+ in the external solution, the channel displayed a rapid flickering behavior. At higher concentrations of divalent cations, the transitions were too rapid to be fully resolved and appeared as a reduction in mean unitary single-channel current amplitude. 3. This effect was voltage dependent, and on analysis was shown to be due to an open channel block by divalent ions. In the case of Mg2+, the block increased steadily with hyperpolarization. In contrast, for Ca2+ the block first increased with hyperpolarization and then decreased with further hyperpolarization beyond -70 mV, providing evidence for Ca2+ permeation of this channel. 4. This block is similar to that seen in voltage-gated calcium channels. Additionally, the cyclic nucleotide-gated channel shows some pharmacological similarities with L-type calcium channels, including a novel block of the cyclic nucleotide channel by nifedipine (50 microM). 5. Our results indicate that the sensory generator current simultaneously depends on the presence of the second messenger and on the membrane potential of the olfactory neuron.

scholarly journals Divalent Cation Interactions with Light-Dependent K Channels

1999 ◽  
Vol 114 (5) ◽  
pp. 653-672 ◽  
Author(s):  
Enrico Nasi ◽  
Maria del Pilar Gomez
Keyword(s):  
Binding Site ◽  
Single Channel ◽  
Divalent Cations ◽  
Light Response ◽  
Relaxation Methods ◽  
Physiological Conditions ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Single Binding Site

The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca2+ and Mg2+ that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca2+ than Mg2+ (K1/2 ≈ 16 and 61 mM, respectively, at Vm = −30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca2+ block is consistent with a single binding site located at an electrical distance of δ ≈ 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (τ = 5–20 ms) that disappears on removal of Ca2+ and Mg2+ and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca2+ than with Mg2+, suggesting that the process is dominated by the “on” rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

scholarly journals Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

2002 ◽  
Vol 120 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Ion Channels ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Acid Sensing Ion Channels ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetics Of ◽  
Channel Properties

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small.

scholarly journals The weak voltage dependence of pannexin 1 channels can be tuned by N-terminal modifications

2018 ◽  
Vol 150 (12) ◽  
pp. 1758-1768 ◽  
Author(s):  
Kevin Michalski ◽  
Erik Henze ◽  
Phillip Nguyen ◽  
Patrick Lynch ◽  
Toshimitsu Kawate
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Atp Release ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Pannexin 1 ◽  
Voltage Dependent ◽  
Gated Channel

Pannexins are a family of ATP release channels important for physiological and pathological processes like blood pressure regulation, epilepsy, and neuropathic pain. To study these important channels in vitro, voltage stimulation is the most common and convenient tool, particularly for pannexin 1 (Panx1). However, whether Panx1 is a voltage-gated channel remains controversial. Here, we carefully examine the effect of N-terminal modification on voltage-dependent Panx1 channel activity. Using a whole-cell patch-clamp recording technique, we demonstrate that both human and mouse Panx1, with their nativeN termini, give rise to voltage-dependent currents, but only at membrane potentials larger than +100 mV. This weak voltage-dependent channel activity profoundly increases when a glycine–serine (GS) motif is inserted immediately after the first methionine. Single-channel recordings reveal that the addition of GS increases the channel open probability as well as the number of unitary conductance classes. We also find that insertions of other amino acid(s) at the same position mimics the effect of GS. On the other hand, tagging the N terminus with GFP abolishes voltage-dependent channel activity. Our results suggest that Panx1 is a channel with weak voltage dependence whose activity can be tuned by N-terminal modifications.

scholarly journals IKs ion-channel pore conductance can result from individual voltage sensor movements

2019 ◽  
Vol 116 (16) ◽  
pp. 7879-7888 ◽  
Author(s):  
Maartje Westhoff ◽  
Jodene Eldstrom ◽  
Christopher I. Murray ◽  
Emely Thompson ◽  
David Fedida
Keyword(s):  
Single Channel ◽  
Voltage Sensor ◽  
Open Probability ◽  
Voltage Sensors ◽  
Repolarization Reserve ◽  
Accessory Subunits ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Open States ◽  
Channel Currents

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance–voltage (G–V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

scholarly journals The Na+/Ca2+ Exchanger Inhibitor, KB-R7943, Blocks a Nonselective Cation Channel Implicated in Chemosensory Transduction

2009 ◽  
Vol 101 (3) ◽  
pp. 1151-1159 ◽  
Author(s):  
A. Pezier ◽  
Y. V. Bobkov ◽  
B. W. Ache
Keyword(s):  
Transient Receptor Potential ◽  
Single Channel ◽  
Receptor Potential ◽  
Trpc Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Olfactory Transduction ◽  
Voltage Dependent ◽  
Chemosensory Transduction ◽  
Transient Receptor

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na+/Ca2+ exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na+/Ca2+ exchanger.

Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

2007 ◽  
Vol 293 (1) ◽  
pp. F236-F244 ◽  
Author(s):  
Ling Yu ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Heavy Metal ◽  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Epithelial Cell Line ◽  
Open Probability ◽  
Renal Epithelial Cell ◽  
Histidine Residues ◽  
Voltage Dependent ◽  
Renal Epithelial Cell Line

To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels ( N) × open probability ( Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2570-2575 ◽  
Author(s):  
L. S. Premkumar ◽  
P. W. Gage
Keyword(s):  
Potassium Channels ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Kinetic Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cultured Hippocampal Neurons

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells

1987 ◽  
Vol 253 (1) ◽  
pp. H210-H214
Author(s):  
M. Horie ◽  
H. Irisawa
Keyword(s):  
Guinea Pig ◽  
Action Potentials ◽  
Single Channel ◽  
Outward Current ◽  
Atrial Cells ◽  
Voltage Dependent ◽  
K Current ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Low K

Rectifying properties of the acetylcholine (ACh)-sensitive K+ channels were studied using a patch-clamp method in single atrial cells prepared enzymatically from adult guinea pig hearts. In the presence of micromolar concentration of nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue 5'-guanylylimidodiphosphate (GppNHp) and the absence of Mg2+ at the inner surface of patch membrane [( Mg2+]i), the channel activity recovered in inside-out patch condition. The single channel conductance became ohmic between -80 and +80 mV (symmetrical 150 mM K+ solutions). The rapid relaxation of outward single channel currents was disclosed on a depolarization. [Mg2+]i blocked the outward current through the channel dose- and voltage-dependently and also induced a dose-dependent increase in the channel activation. The apparent paradoxical role of [Mg2+]i is important for the cholinergic control in the heart; voltage-dependent Mg block ensures a low K+ conductance of cell membrane at the plateau of action potentials during the exposure to ACh, thereby slowing the heart rate without unfavorable shortening of the action potentials.

scholarly journals Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1

2002 ◽  
Vol 120 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Lai-Hua Xie ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Single Channel ◽  
Quantitative Model ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Surface Charges ◽  
Open Probability ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inside Out

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

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