SEARCH · PubMed Health

Results for “patch clamp”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 829 records · Page 46Linked to original sources

PubMed1997

Imaging excised apical plasma membrane patches of MDCK cells in physiological conditions with atomic force microscopy.

We combined the patch-clamp technique with atomic force microscopy (AFM) to visualize plasma membrane proteins protruding from the extracellular surface of cultured kidney cells (MDCK cells). To achieve molecular resolution, patches were mechanically isolated from whole MDCK cells by applying the patch-clamp technique. The excised inside-out patches were transferred on freshly cleaved mica and imaged with the AFM in air and under physiological conditions (i. e. in fluid). Thus, the resolution could be increased considerably (lateral and vertical resolutions 5 and 0.1 nm, respectively) as compared to experiments on intact cells, where plasma membrane proteins were hardly detectable. The apical plasma membrane surface of the MDCK cells showed multiple protrusions which could be identified as membrane proteins through the use of pronase. These proteins had a density of about 90 per micron(2), with heights between 1 and 9 nm, and lateral dimensions of 20-60 nm. Their frequency distribution showed a peak value of 3 nm for the protein height. A simplified assumption - modelling plasma membrane proteins as spherical structures protruding from the lipid bilayer - allowed an estimation of the possible molecular weights of these proteins. They range from 50 kDa to 710 kDa with a peak value of 125 kDa. We conclude that AFM can be used to study the molecular structures of membranes which were isolated with the patch-clamp technique. Individual membrane proteins and protein clusters, and their arrangement and distribution in a native plasma membrane can be visualized under physiological conditions, which is a first step for their identification.

Animals↗

PubMed1996

Adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor in hippocampal neurons.

The effects of adenosine on hippocampal neurons were examined by patch-clamp recording and Ca2+ imaging using fura-2 fluorescence. In the whole-cell patch-clamp configuration, adenosine evoked outwardly rectifying K+ currents in a dose-dependent manner. These currents were not inhibited by a nonselective P1 purinoceptor antagonist or selective adenosine A1, A2A receptor antagonists and moreover, selective adenosine A1, A2A receptor agonists evoked no current. In contrast, P2 purinoceptor agonists produced similar outward currents with the order of potency: ADP > or = 2-methylthio ATP > ATP > adenosine >> AMP. No response was obtained to UTP, alpha, beta-methylene ATP or beta, gamma-methylene ATP. The intracellular perfusion of a broad G-protein inactivator, guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), abolished adenosine-evoked currents, whereas a Gi/Go-protein inhibitor, pertussis toxin, had no effect. Furthermore, the currents were blocked by a phospholipase C inhibitor, neomycin, or specific protein kinase C inhibitors, GF109203X (bisindolyl maleimide, C25H24N4O2) and protein kinase C inhibitor peptide. In the cell-attached patch-clamp configuration, adenosine elicited single-channel currents with two major kinds of slope conductances. Likewise, application of adenosine outside the patch electrode again produced single-channel currents with same conductances. A potent protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), induced single-channel currents in a fashion that mimics the effect of adenosine. The evoked currents were blocked by GF109203X. In addition, adenosine enhanced intracellular free Ca2+ concentration ([Ca2+]i). This [Ca2+]i increase was inhibited by GDP beta S or neomycin, but was not affected by pertussis toxin. These results, thus, suggest that adenosine activates the K+ channel and enhances cytosolic Ca2+ release via a P2Y purinoceptor linked to a pertussis toxin-insensitive G-protein, which is involved in a phospholipase C-mediated phospholipid-signaling pathway.

Adenosine↗

PubMed1991

Effects of hypothalamic peptides on electrical activity and membrane currents of 'patch perforated' clamped GH3 anterior pituitary cells.

'Perforated-patch' recordings of rat anterior pituitary GH3 cells allow long and stable monitoring of electrical activity and membrane currents. Under current clamp conditions, the biphasic effect of thryotropin releasing hormone (TRH) consisting of a transient hyperpolarization followed by a longer phase of increased action potential frequency is fully preserved. Somatostatin suppresses action potential activity and antagonizes the second phase of enhanced spiking caused by TRH. Voltage clamp records of isolated currents indicate that TRH affects calcium-dependent potassium currents, but does not alter either voltage-dependent potassium or calcium currents at times and concentrations at which the electrical activity is increased.

Animals↗

PubMed1997

Cyclic nucleotide-gated cation channels mediate sodium absorption by IMCD (mIMCD-K2) cells.

The inner medullary collecting duct cell line, mIMCD-K2, absorbs Na+ by an amiloride-sensitive, electrogenic mechanism. The goal of the present study was to characterize the amiloride-sensitive, Na+ -conducting channels responsible for electrogenic Na+ absorption. To this end, we measured Na+ currents in single cells with the patch-clamp technique and Na+ currents across monolayers mounted in Ussing-type chambers. In whole cell patch-clamp experiments, amiloride-sensitive, inward Na+ currents were mediated by nonselective cation channels. In single-channel patch-clamp experiments, amiloride- and guanosine 3',5'-cyclic monophosphate (cGMP)-sensitive, 20-pS nonselective cation channels (i.e., CNG channels) were identified in the apical membrane. CNG channels were inhibited by amiloride, diltiazem, ethylisopropylamiloride (EIPA), and 8-bromo-cGMP and were permeable to Ca2+ and Mg2+. Epithelial Na+ channels were never observed in whole cell or single-channel recordings. Na+ absorption across confluent monolayers was inhibited with a rank order potency of benzamil > amiloride > phenamil >> EIPA > diltiazem. Our data are most consistent with the view that CNG channels mediate electrogenic Na+ absorption across mIMCD-K2 cells.

Amiloride↗

PubMed2006

Establishment of cell-free electrophysiology for ion transporters: application for pharmacological profiling.

Ion transporters are emerging targets of increasing importance to the pharmaceutical industry because of their relevance to a wide range of numerous indications of cardiovascular, metabolic, and inflammatory diseases. However, traditional ion transporter assay technologies using radioactive or fluorescent ligands and substrates or manual patch clamping suffer from several problems: limited sensitivity and robustness, significant numbers of false positives and false negatives, and cost. The authors describe a novel method for the measurement of ion transporters using cell-free electrophysiology based on the SURFE (2) R (surface electrogenic event reader) technology platform. The main advantages of the method described here are high sensitivity and simple handling. Material for assays is mainly a simple membrane preparation, which can be stored over weeks and months. Thus, the application of the method does not depend on a permanently running cell-culture lab. The application of the technology itself uses a bench-top system and chips loaded with membrane fragments. The SURFE (2) R technology was used to establish an Na+/Ca2+-exchanger assay. The assay performance, as judged by the Z' value of 0.73 and the signal-to-background ratio of 7.6, suggests that this is a reliable and robust assay. The authors compared the technology with patch-clamp experiments: The measurement of activity of 17 different inhibitors and the determination of an IC (50)value indicated a good correlation between SURFE (2) R technology and patch clamp results. Using the SURFE (2) R technology, results were obtained with 20 times higher throughput and required less-qualified personnel compared with manual patch clamping.

Animals↗

PubMed1995

Functional expression of bacteriorhodopsin in oocytes allows direct measurement of voltage dependence of light induced H+ pumping.

We report on the first successful expression of the light driven H+ pump, bacteriorhodopsin, into the plasma membrane of oocytes from Xenopus laevis. The light induced photocurrents which reflect the pumping of H+ by BR were analysed under voltage clamp conditions. At least 100 active BR molecules per microns 2 were expressed in the plasma membrane so that both the voltage clamp and giant patch clamp method could be applied. We show that H+ pumping by BR is modulated by the membrane potential, i.e. the pump current shows strong voltage dependence in the range measured between -165 mV to +60 mV.

Animals↗

PubMed1997

Identification of functionally important regions of the muscular chloride channel CIC-1 by analysis of recessive and dominant myotonic mutations.

Mutations in the muscular voltage-dependent Cl-channel, CIC-1, lead to recessive and dominant myotonia. Here we analyse the effects of one dominant (G200R) and three recessive (Y150C, Y261C, and M485V) mutations after functional expression in Xenopus oocytes. Glycine 200 is a highly conserved amino acid located in a conserved stretch in the putatively cytoplasmic loop between domains D2 and D3. Similar to several other dominant mutations the amino acid exchange G200R leads to a drastic shift by approximately 65 mV of the open probability curve to more positive voltages. As explored by co-expression studies, the shift is intermediate in heteromeric mutant/WT channels. Open channel properties such as single channel conductance, rectification or ion selectivity are not changed. Thus we identified a new region of the CIC-1 protein in which mutations can lead to drastic shifts of the voltage dependence. The recessive mutation M485V, which is located in a conserved region at the beginning of domain D10, leads to a drastic reduction of the single channel conductance from 1.5 pS for WT to approximately 0.3 pS. In addition, the mutant is strongly inwardly rectifying and deactivates incompletely at negative voltages. Ion-selectivity, however, is unchanged. These electrophysiological properties fully explain the recessive phenotype of the mutation and identify a new region of the protein that is involved in ion permeation and gating of the CIC-1 channel. The other two recessive mutations (Y150C and Y261C) had been found in a compound heterozygous patient. Surprisingly, expression of these mutants in oocytes yielded currents indistinguishable from WT CIC-1 when explored by two-electrode voltage clamp recording and patch clamping (either singly or both mutations co-expressed). Other mechanisms that are not faithfully represented by the Xenopus expression system must therefore be responsible for the myotonic symptoms associated with these mutations.

Amino Acid Sequence↗

PubMed2004

Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors.

NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose-response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30- to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.

Animals↗

PubMed1998

The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels.

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.

Adenosine Triphosphate↗

PubMed2003

KCNE2 modulates current amplitudes and activation kinetics of HCN4: influence of KCNE family members on HCN4 currents.

The HCN4 gene encodes a hyperpolarization-activated cation current contributing to the slow components of the pacemaking currents I(f) in the sinoatrial node and I(h) or I(q) in the thalamus. Heterologous expression studies of individual HCN channels have, however, failed to reproduce fully the diversity of native I(f/h/q) currents, suggesting the presence of modulating auxiliary subunits. Consistent with this is the recent description of KCNE2, which is highly expressed in the sinoatrial node, as a beta-subunit of rapidly activating HCN1 and HCN2 channels. To determine whether KCNE2 can also modulate the slow component of native I(f/h/q) currents, we co-expressed KCNE2 with HCN4 in Xenopus oocytes and in Chinese hamster ovary (CHO) cells and analysed the resulting currents using two-electrode voltage-clamp and patch-clamp techniques, respectively. In both cell types, co-expressed KCNE2 enhanced HCN4-generated current amplitudes, slowed the activation kinetics and shifted the voltage for half-maximal activation of currents to more negative voltages. In contrast, the related family members KCNE1, KCNE3 and KCNE4 did not change current characteristics of HCN4. Consistent with these electrophysiological results, the carboxy-terminal tail of KCNE2, but not of other KCNE subunits, interacted with the carboxy-terminal tail of HCN4 in yeast two-hybrid assays. KCNE2, by modulating I(f) or I(h) currents, might thus contribute to the electrophysiological diversity of known pacemaking currents in the heart and brain.

Animals↗

PubMed2004

Potent activation of the human tandem pore domain K channel TRESK with clinical concentrations of volatile anesthetics.

The tandem pore domain K channel family mediates background K currents present in excitable cells. Currents passed by certain members of the family are enhanced by volatile anesthetics, thus suggesting a novel mechanism of anesthesia. The newest member of the family, termed TRESK (TWIK [tandem pore domain weak inward rectifying channel]-related spinal cord K channel), has not been studied for anesthetic sensitivity. We isolated the coding sequence for TRESK from human spinal cord RNA and functionally expressed it in Xenopus oocytes and transfected COS-7 cells. With both whole-cell voltage-clamp and patch-clamp recording, TRESK currents increased up to three-fold by clinical concentrations of isoflurane, halothane, sevoflurane, and desflurane. Nonanesthetics (nonimmobilizers) had no effect on TRESK. Various IV anesthetics, including etomidate, thiopental, and propofol, have a minimal effect on TRESK currents. Amide and ester local anesthetics inhibit TRESK in a concentration-dependent manner but at concentrations generally larger than those that inhibit other tandem pore domain K channels. We also determined that TRESK is found not only in spinal cord, but also in human brain RNA. These results identify TRESK as a target of volatile anesthetics and suggest a role for this background K channel in mediating the effects of inhaled anesthetics in the central nervous system.

Anesthetics, Inhalation↗

PubMed2003

Mutations at the GABA receptor selectivity filter: a possible role for effective charges.

An important feature of ligand-gated ion channels is their exquisite ability to discriminate between ions. Still, little is known about the mechanisms underlying, or structural determinates of, this ability. We examined the structural elements underlying the ionic selectivity of rho1 GABA receptors expressed in Xenopus oocytes and human embryonic kidney cells using site-directed mutagenesis and two-electrode voltage-clamp or patch-clamp techniques. The wild-type GABA receptor was chloride selective, with a small but significant permeability to potassium (PNa+ : PK+ : PCl- = 0 : 0.03 :1). Mutation of an alanine to glutamate at position 291 (thought to be located at the intracellular end of the second transmembrane domain), formed a channel that exhibited little discrimination among ions (0.70:0.87:1), while deletion of a neighbouring proline (290) was chloride selective, but had elevated cation permeabilities compared to the wild-type channel (0.12 : 0.14 : 1). Together, the two mutations (DeltaP290/A291E) caused a reversal of selectivity (2.72 : 3.59 : 1). We also examined the effects of neutralizing and reversing the charge of the adjacent, and highly conserved, arginine. Mutation of the neighbouring arginine to glutamate (R292E) increased the cation permeability similar to the DeltaP290/A291E double mutant (2.4 : 3.0 : 1), whereas neutral mutations at this position (R292M or R292C) retained chloride selectivity (0 : 0.11 : 1.0 and 0 : 0.14 : 1.0, respectively). Our experiments suggest that the effective charge near the presumed intracellular mouth of the pore is critical for ionic selectivity.

Algorithms↗

PubMed2000

Neuroreceptors and ion channels as the basis for drug action: past, present, and future.

This article summarizes the development of cellular neuropharmacology and neurotoxicology, based primarily on my own research. The progress of this field depends at least in part on the theoretical and technological developments of excitable cell physiology, biophysics, and biochemistry. First, a brief historical development is described. Second, my earlier studies of the mechanism of action of insecticides on the nervous system are introduced. The most significant is the early discovery of the increase in depolarizing after-potential caused by DDT and pyrethroids. This laid the foundation of subsequent analyses of sodium channel modulation as the major mechanism of action of DDT/pyrethroids. Third, my initial contributions to cellular neuropharmacology are described. The discovery of the potent and selective block of sodium channels by tetrodotoxin aroused interest not only in using this toxin and other chemicals as useful laboratory tools but also in studying receptors/channels as important targets of various drugs. Using internally perfused squid giant axons, pioneering studies of local anesthetic action led to the conclusion that these anesthetics block the sodium channel from inside the nerve membrane in the cationic form. Fourth, a few examples of my more recent studies using voltage-clamp and patch-clamp techniques are described. Pyrethroid modulation of sodium channels was analyzed in great detail, including single-channel kinetics, toxicity amplification from channels to animal behaviors, temperature dependence, selective toxicity, and vitamin E antagonism. The neuroprotective drug riluzole blocked sodium channels and high-voltage-activated calcium channels, thereby preventing excess stimulation of N-methyl-D-aspartate receptors and massive influx of calcium, thereby retarding spread of infarction in the brain. Neuronal nicotinic acetylcholine receptors have received much attention recently, and I launched an extensive study of the mechanism whereby alcohols and general anesthetics modulate their activity. Ethanol potently stimulates the alpha-bungarotoxin-insensitive, alpha4beta2-type acetylcholine receptors, thereby causing release of various transmitters; this leads to a cascade of multisynaptic events and behavioral changes. Inhalational general anesthetics augment the activity of gamma-aminobutyric acid(A) receptors and inhibit the activity of alpha4beta2-type acetylcholine receptors, causing a variety of clinical syndromes. Fifth, one of the possible future directions of cellular neuropharmacology and neurotoxicology is discussed. Emphasis is placed on the three-dimensional structure-activity relationship, in particular how changes in the molecular structure of drugs and receptors/channels result in kinetic changes in the function of receptors/channels.

Anesthetics, Local↗

PubMed2002

Remodelling inactivation gating of Kv4 channels by KChIP1, a small-molecular-weight calcium-binding protein.

Calcium-binding proteins dubbed KChIPs favour surface expression and modulate inactivation gating of neuronal and cardiac A-type Kv4 channels. To investigate their mechanism of action, Kv4.1 or Kv4.3 were expressed in Xenopus laevis oocytes, either alone or together with KChIP1, and the K+ currents were recorded using the whole-oocyte voltage-clamp and patch-clamp methods. KChIP1 similarly remodels gating of both channels. At positive voltages, KChIP1 slows the early phase of the development of macroscopic inactivation. By contrast, the late phase is accelerated, which allows complete inactivation in < 500 ms. Thus, superimposed traces from control and KChIP1-remodelled currents crossover. KChIP1 also accelerates closed-state inactivation and recovery from inactivation (3- to 5-fold change). The latter effect is dominating and, consequently, the prepulse inactivation curves exhibit depolarizing shifts (DeltaV = 4-12 mV). More favourable closed-state inactivation may also contribute to the overall faster inactivation at positive voltages because Kv4 channels significantly inactivate from the preopen closed state. KChIP1 favours this pathway further by accelerating channel closing. The peak G-V curves are modestly leftward shifted in the presence of KChIP1, but the apparent 'threshold' voltage of current activation remains unaltered. Single Kv4.1 channels exhibited multiple conductance levels that ranged between 1.8 and 5.6 pS in the absence of KChIP1 and between 1.9 and 5.3 pS in its presence. Thus, changes in unitary conductance do not contribute to current upregulation by KChIP1. An allosteric kinetic model explains the kinetic changes by assuming that KChIP1 mainly impairs open-state inactivation, favours channel closing and lowers the energy barrier of closed-state inactivation.

Animals↗

PubMed2006

Open probability of the epithelial sodium channel is regulated by intracellular sodium.

The regulation of epithelial Na(+) channel (ENaC) activity by Na(+) was studied in Xenopus oocytes using two-electrode voltage clamp and patch-clamp recording techniques. Here we show that amiloride-sensitive Na(+) current (I(Na)) is downregulated when ENaC-expressing cells are exposed to high extracellular [Na(+)]. The reduction in macroscopic Na(+) current is accompanied by an increase in the concentration of intracellular Na(+) ([Na(+)](i)) and is only slowly reversible. At the single-channel level, incubating oocytes in high-Na(+) solution reduces open probability (P(o)) approximately twofold compared to when [Na(+)] is kept low, by increasing mean channel closed times. However, increasing P(o) by introducing a mutation in the beta-subunit (S518C) which, in the presence of [2-(trimethylammonium) ethyl] methane thiosulfonate (MTSET), locks the channel in an open state, could not alone abolish the downregulation of macroscopic current measured with exposure to high external [Na(+)]. Inhibition of the insertion of new channels into the plasma membrane using Brefeldin A revealed that surface channel lifetime is also markedly reduced under these conditions. In channels harbouring a beta-subunit mutation, R564X, associated with Liddle's syndrome, open probability in both high- and low-Na(+) conditions is significantly higher than in wild-type channels. Increasing the P(o) of these channels with an activating mutation abrogated the difference in macroscopic current observed between groups of oocytes incubated in high- and low-Na(+) conditions. These findings demonstrate that reduction of ENaC P(o) is a physiological mechanism limiting Na(+) entry when [Na(+)](i) is high.

Animals↗

PubMed2000

Pharmacological activation of cloned intermediate- and small-conductance Ca(2+)-activated K(+) channels.

We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca(2+)-activated K(+) channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca(2+)-dependent K(+) channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal (K(1/2)) values of 84 microM and 98 microM, respectively. Similarly, CZ activated rSK2 with a K(1/2) of 87 microM. In the absence of CZ, the Ca(2+)-dependent activation of hIK1 was best fit with a K(1/2) of 700 nM and a Hill coefficient (n) of 2.0. rSK2 was activated by Ca(2+) with a K(1/2) of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K(1/2) or n for Ca(2+)-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca(2+) concentrations (V(max)). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on tau(o1), tau(o2), or tau(c1), whereas tau(c2) and tau(c3) were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.

Adenosine Triphosphate↗

PubMed1997

Diversity of channels generated by different combinations of epithelial sodium channel subunits.

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: alpha, beta, and gamma; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na+ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of alpha with beta and of alpha with gamma in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that alpha beta channels differ from alpha gamma channels in the following functional properties: (a) alpha beta channels expressed larger Na+ than Li+ currents (INa+/ILi+ 1.2) whereas alpha gamma channels expressed smaller Na+ than Li+ currents (INa+/ILi+ 0.55); (b) the Michaelis Menten constants (Km of activation of current by increasing concentrations of external Na+ and Li+ of alpha beta channels were larger (Km > 180 mM) than those of alpha gamma channels (Km of 35 and 50 mM, respectively); (c) single channel conductances of alpha beta channels (5.1 pS for Na+ and 4.2 pS for Li+) were smaller than those of alpha gamma channels (6.5 pS for Na+ and 10.8 pS for Li+); (d) the half-inhibition constant (Ki) of amiloride was 20-fold larger for alpha beta channels than for alpha gamma channels whereas the Ki of guanidinium was equal for both alpha beta and alpha gamma. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the gamma subunit and the carboxy terminus of the beta subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the beta subunit, was identified as the domain conferring lower amiloride affinity to the alpha beta channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits alpha with beta or gamma contribute to these binding sites. Finally, we show that the most likely stoichiometry of alpha beta and alpha gamma channels is 1 alpha: 1 beta and 1 alpha: 1 gamma, respectively.

Amiloride↗

PubMed2006

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.

4-Aminopyridine↗