Cell Voltage-clamp Studies Research Articles

A Heterozygous Missense hERG Mutation Associated with Early Repolarization Syndrome

Background/Aims: Early repolarization syndrome (ERS) has been recently recognized as early repolarization pattern with idiopathic ventricular fibrillation. However, the genetic background of ERS has not been fully understood. Methods: A Chinese family with sudden cardiac death associated with ERS was investigated. Direct sequencing of ERS susceptibility genes was performed on the proband and family members. Whole-cell patch-clamp methods were used to characterize the mutant channel expressed in HEK 293 cells. Results: One missense mutation (p. K801T) was found in the hERG (KCNH2 gene) by the direct sequencing of candidate genes. Whole cell voltage clamp studies of the K801T mutation in HEK 293 cells demonstrated a 1.5-fold increase in maximum steady state current (37.2±7.3 vs 20.3±4.4 pA/pF) that occurred at a 20 mV more positive potential compared to the wild type channels. The voltage dependence of inactivation was significantly shifted in the positive voltage direction (WT -59.5±1.4 vs K801T -44.3±1.2 mV). Kinetic analysis revealed slower inactivation rates of K801T, but faster rates of activation and deactivation. The hERG channel blockers tested inhibited K801T-hERG channel in concentration response, and the potencies of these drugs can be rank-ordered as follows: quinidine> disopyramide> sotalol> flecainide. Conclusion: Our study indicated that the K801T mutation caused the gain of function of hERG channels that may account for the clinical phenotype of ERS. Quinidine and disopyramide could improve the function of K801T-hERG mutant channel, and may be therapeutic options for patients with the K801T hERG mutation.

The Role of Toll‐Like Receptor 4 in Enteric Glia

Toll‐like receptors (TLRs) are a class of pattern recognition receptors that play an important role in mediating inflammatory responses to pathogens. TLR4 recognizes lipopolysaccharide (LPS), a membrane component of gram negative bacteria. In the GI tract, neuronal integrity may be dependent on basal activation of TLR4 receptors suggesting a protective role for TLR4. A compromised gut epithelial barrier leads to enhanced entry of bacterial proteins into the lamina propria, leading to neuronal toxicity. Thus pro‐survival vs pro‐toxic effects of LPS may be concentration‐dependent. Enteric glia are important for the maintenance of neuronal integrity. We determined the expression of pro‐inflammatory cytokines in rat enteric glia cells in response to low and high concentrations of LPS. LPS dose‐dependently increased mRNA expression of IL‐6 and TNF‐α with 0.1 μg/ml inducing a 25‐fold and 10‐fold IL‐6 and TNF‐α mRNA expression respectively as compared to untreated glia. A 100‐fold higher concentration of LPS (1‐100 μg/ml) also induced ATP release indicative of cell damage. Whole cell voltage clamp studies showed that this high concentration of LPS increased ATP‐induced inward currents with a corresponding increase in P2X1,2,3,4,and 7 receptor mRNA expression up to 50‐fold as compared to untreated glia. Thus low concentrations of LPS can induce cytokine release whereas high concentrations of LPS induce ATP release which increases P2X receptor activity and further enhances the release of pro‐inflammatory cytokines. Understanding the role of P2X in LPS induced enteric glia activation will point to new targets to control gut bacterial inflammation while still maintaining the beneficial effects of basal TLR4 signaling. Supported by NIH DK046367, DA024009.

A novel mutation in the KCNH2 gene associated with short QT syndrome

A gain of function mutation N588K in the KCNH2 gene that encodes HERG channels has been shown to underlie the SQT1 form of short QT syndrome (SQTS). We describe a different mutation in the KCNH2 gene in a Chinese family with clinical evidence of SQTS. A Chinese family with a markedly short QT interval (QTc=316±9ms, n=4) and a strong family history of sudden death was investigated. Analysis of candidate genes contributing to ventricular repolarization identified a C1853T mutation in the KCNH2 gene coding for the HERG channel, resulting in an amino acid change (T618I) that was found to 100% co-segregate with the SQTS phenotype (n=4). Whole cell voltage clamp studies of the T618I mutation in HEK-cells demonstrated a 6-fold increase in maximum steady state current (146.1±16.7 vs 23.8±5.5pA/pF) that occurred at a 20mV more positive potential compared to the wild type channels. The voltage dependence of inactivation was significantly shifted in the positive voltage direction (WT −78.6±6.8 vs T618I −29.3±1.7mV). Kinetic analysis revealed slower inactivation rates of T618I but faster rates of recovery from inactivation. Quinidine (5μM) and sotalol (500μM) had similar inhibitory effects on steady currents measured at +20mV in WT and T618I but were less effective in inhibiting tail currents of mutant channels. The altered function of T618I-HERG channels suggests that this mutation in the KCNH2 gene is responsible for the SQTS phenotype in this family. Both quinidine and sotalol may be therapeutic options for patients with the T618I HERG mutation.

Identification and Modulation of Voltage-Gated Ca2+Currents in Zebrafish Rohon-Beard Neurons

Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca(2+) channels (VGCCs) are especially important as Ca(2+) serves as both a charge carrier and second messenger. Zebrafish (Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, (Isl2b:EGFP)(ZC7), were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca(2+) current (HVA- and LVA-I(Ca), respectively). Ni(+)-sensitive LVA-I(Ca) occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive Ca(V)2.2 (N-type) Ca(2+) channels underlie the vast majority (90%) of HVA-I(Ca). To identify G protein coupled receptors (GPCRs) that modulate HVA-I(Ca), a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of I(Ca) appears to be transduced primarily via a cholera toxin-sensitive Gα subunit. These results provide the basis for using the zebrafish model system to understanding Ca(2+) channel function, and in turn, how Ca(2+) channels contribute to mechanosensory function.

Abstract 138: Transient Receptor Potential Channel C1 Deficiency Protects the Murine Heart from Pressure Overload

Transient receptor potential canonical (TRPC) channels are non-selective cation channels that are activated in response to G-protein coupled receptor activation, depletion of internal stores and mechanical stretch. Recent reports suggest that cardiac TRPC channels mediate calcineurin dependent cardiac hypertrophy, yet few details exist as to the mechanism for activation of these channels. Here, we provide evidence that TRPC1 channels are the dominant TRPC channel in mouse cardiomyocytes and cardiac TRPC1 protein expression is augmented by seven fold following thoracic aortic banding (TAC). In addition, we provide the first loss of function studies to show that mice lacking TRPC1 channels developed significantly less cardiac hypertrophy following pressure overload induced by thoracic aortic banding suggesting that TRPC1 may confer deleterious calcium entry. Whole cell voltage clamp studies of isolated adult cardiomyocytes reveal a non-selective cation current that is induced by pressure overload that is absent in TRPC1−/− cardiomyocytes and in which TRP blockers such as gadolinium, 2-amino biphenyl boric acid and SKF96365 inhibit the TAC induced current. Finally, neonatal cardiomyocytes lacking functional TRPC1 display reduced TRPC current in response to cell stretch or angiotensin-II; the functional consequence of which includes reduced calcium oscillation frequency and reduced BNP expression. These results provide the first loss of function evidence for TRPC1 channels in cardiac hypertrophy and implicate TRPC1 as a stretch activated channel.

Nitroglycerin‐Induced Relaxation is Mediated by BKCa Channels in Nitrate Tolerant Arteries

Numerous studies have examined the cause(s) of nitrate tolerance, but remarkably little is known about possible adaptive processes that may occur in nitrate-tolerant blood vessels. We tested the hypothesis that nitrate tolerance induces a compensatory response involving K channels in nitroglycerin-induced smooth muscle relaxation. Rats were treated with placebo or NTG (0.6 mg/hr) patches for 3 days. Thoracic aortae were removed, cut into rings, and suspended in organ baths. Relaxation responses to NTG (10−9 to 10−5 M) were obtained in the absence and presence of several K channel blockers. Nitrate tolerance was evident by a >7-fold rightward shift in the NTG concentration-response curve in tissues from rats treated with NTG patches as compared with placebo. Iberiotoxin (10−7 M), a selective blocker of BKCa, had no effect on NTG-induced relaxation of rings from nontolerant rats, but caused a further rightward shift (>7-fold) in the concentration-response curve to NTG in rings from tolerant rats. Selective blockers of other K channels, including glyburide (10−6 M), apamin (10−6 M), and 4-aminopyridine (10−3 M) had no effect on responses to NTG in either nontolerant or tolerant rings. Whole cell voltage-clamp studies from placebo and NTG-treated rat aortic myocytes indicated that NTG (10 μM) significantly increased BKCa current density in tolerant aortic myocytes at +80 mV (22.6 ± 3.02 and 52.5 ± 5.52 pA/pF, control and tolerant respectively). The data suggests that in nontolerant arteries, NTG-induced relaxation is independent of BKCa channel activation. By contrast, prolonged in vivo treatment with NTG results in a major role for BKCa in NTG-induced relaxation of nitrate tolerant arteries.

Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants

Neonicotinoid insecticides are compounds acting agonistically on insect nicotinic acetylcholine receptors (nAChR). They are especially active on hemipteran pest species such as aphids, whiteflies, and planthoppers, but also commercialized to control many coleopteran and some lepidopteran pest species. The most prominent member of this class of insecticides is imidacloprid. All neonicotinoid insecticides bind with high affinity ( I 50-values around 1 nM) to [ 3H]imidacloprid binding sites on insect nAChRs. One notable ommission is the neonicotinoid thiamethoxam, showing binding affinities up to 10,000-fold less potent than the others, using housefly head membrane preparations. Electrophysiological whole cell voltage clamp studies using neurons isolated from Heliothis virescens ventral nerve cord showed no response to thiamethoxam when applied at concentrations of 0.3 mM, although the symptomology of poisoning in orally and topically treated noctuid larvae suggested strong neurotoxicity. Other neonicotinoids, such as clothianidin, exhibited high activity as agonists on isolated neurons at concentrations as low as 30 nM. There was no obvious correlation between biological efficacy of thiamethoxam against aphids and lepidopterans and receptor affinity in electrophysiological and binding assays. Pharmacokinetic studies using an LC-MS/MS approach to analyze haemolymph samples taken from lepidopteran larvae revealed that thiamethoxam orally applied to 5th instar Spodoptera frugiperda larvae was rapidly metabolized to clothianidin, an open-chain neonicotinoid. Clothianidin shows high affinity to nAChRs in both binding assays and whole cell voltage clamp studies. When applied to cotton plants, thiamethoxam was also quickly metabolized, with clothianidin being the predominant neonicotinoid in planta briefly after application, as indicated by LC-MS/MS analyses. Interestingly, the N-desmethylated derivative of thiamethoxam, N-desmethyl thiamethoxam, was not significantly produced in either lepidopteran larvae or in cotton plants, although it was often mentioned as a possible metabolite, being nearly as active as imidacloprid. In conclusion, our investigations show that thiamethoxam is likely to be a neonicotinoid precursor for clothianidin.

Mu opiates inhibit long-term potentiation induction in the spinal cord slice.

Long-term potentiation (LTP) involves a prolonged increase in neuronal excitability following repeated afferent input. This phenomenon has been extensively studied in the hippocampus as a model of learning and memory. Similar long-term increases in neuronal responses have been reported in the dorsal horn of the spinal cord following intense primary afferent stimulation. In these studies, we utilized the spinal cord slice preparation to examine effects of the potently antinociceptive mu opioids in modulating primary afferent/dorsal horn neurotransmission as well as LTP of such transmission. Transverse slices were made from the lumbar spinal cord of 10- to 17-day-old rats, placed in a recording chamber, and perfused with artificial cerebrospinal fluid also containing bicuculline (10 microM) and strychnine (1 microM). Primary afferent activation was achieved in the spinal slice by electrical stimulation of the dorsal root (DR) or the tract of Lissauer (LT) which is known to contain a high percentage of small diameter fibers likely to transmit nociception. Consistent with this anatomy, response latencies of LT-evoked field potentials in the dorsal horn were considerably slower than the response latencies of DR-evoked potentials. Only LT-evoked field potentials were found to be reliably inhibited by the mu opioid receptor agonist [D-Ala(2), N-Me-Phe(4), Gly(5)] enkephalin-ol (DAMGO, 1 microM), although evoked potentials from both DR and LT were blocked by the AMPA/kainate glutamate receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione. Moreover repeated stimulation of LT produced LTP of LT- but not DR-evoked potentials. In contrast, repeated stimulation of DR showed no reliable LTP. LTP of LT-evoked potentials depended on N-methyl-D-aspartate (NMDA) receptor activity, in that it was attenuated by the NMDA antagonist APV. Moreover, such LTP was inhibited by DAMGO interfering with LTP induction mechanisms. Finally, in whole cell voltage-clamp studies of Lamina I neurons, DAMGO inhibited excitatory postsynaptic current (EPSC) response amplitudes from LT stimulation-evoked excitatory amino acid release but not from glutamate puffed onto the cell and increased paired-pulse facilitation of EPSCs evoked by LT stimulation. These studies suggest that mu opioids exert their inhibitory effects presynaptically, likely through the inhibition of glutamate release from primary afferent terminals, and thereby inhibit the induction of LTP in the spinal dorsal horn.

Gene expression and function of adenosine A(2A) receptor in the rat carotid body.

The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A(2A) receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A(2A) and A(2B) Ado receptor mRNAs but not the A(1) or A(3) receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A(2A) receptor mRNA. Immunohistochemical studies further showed that the A(2A) receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca(2+) currents and that this inhibition was abolished by the selective A(2A) receptor antagonist ZM-241385. Ca(2+) imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca(2+)](i). Taken together, our findings indicate that A(2A) receptors are present in type I cells and that activation of A(2A) receptors modulates Ca(2+) accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca(2+) homeostasis and cellular excitability during hypoxia.

PKA holoenzyme is functionally coupled to CFTR by AKAPs.

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 +/- 0.12 to 1.23 +/- 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.

Electrophysiological mechanisms by which hypothyroidism delays repolarization in guinea pig hearts.

Thyroid hormone is known to exert important effects on cardiac repolarization, but the underlying mechanisms are poorly understood. We investigated the electrophysiological mechanisms of differences in repolarization between control guinea pigs and hypothyroid animals (thyroidectomy plus 5-propyl-2-thiouracil). Hypothyroidism significantly prolonged the rate-corrected Q-T interval in vivo and action potential duration (APD) of isolated ventricular myocytes. Whole cell voltage-clamp studies showed no change in current density or kinetics of L-type Ca(2+) current, inward rectifier K(+) current, or Na(+) current in hypothyroid hearts. Dofetilide-resistant current (I(Ks)) step current densities were smaller by approximately 65%, and tail current densities were reduced by 80% in myocytes from hypothyroid animals compared with controls. The ratio of delayed rectifier step current at +50 mV to tail current at -40 mV was significantly larger in hypothyroid cells for test pulses from 60- to 4,200-ms duration, reflecting a smaller I(Ks). Dofetilide-sensitive current (I(Kr)) densities were not significantly changed. I(Ks) half-activation voltage shifted to more positive voltages in hypothyroidism (29.5 +/- 2.2 vs. 21.3 +/- 2.7 mV in control, P < 0.01), whereas I(Kr) voltage dependence was unchanged. We conclude that hypothyroidism delays repolarization in the guinea pig ventricle by decreasing I(Ks), a novel and potentially important mechanism for thyroid regulation of cardiac electrophysiology.

Hydrogen peroxide activates glibenclamide-sensitive K+ channels in LLC-PK1 cells.

Oxidant-induced damage has been implicated in the pathogenesis of several forms of cellular injury. The present study employed patch-clamp methods to determine if oxidant stress leads to activation of plasma membrane K+ channels in the renal epithelial LLC-PK1 cell line. Exposure of cells to H2O2 (0.1 to 5 mM) induced a rapid (within 5-10 min), dose-dependent membrane hyperpolarization. Perforated patch whole cell voltage-clamp studies were performed to determine the ion selectivity of the currents underlying this H2O2-induced cellular hyperpolarization. H2O2 (5 mM) produced a sixfold increase in the whole cell conductance. The reversal potential of the H2O2-induced current was consistent with a K+-selective conductance. This current was blocked almost completely by 5 mM barium and 500 microM glibenclamide but only partially by 15 mM tetraethylammonium. Exposure of LLC-PK1 cells to 5 mM H2O2 reduced cell ATP content by 70%. To evaluate more directly the role of ATP depletion in the activation of K+ channels, conventional whole cell patch-clamp studies were performed. Inclusion of ATP in the pipette solution prevented H2O2-induced activation of the K+ conductance. These findings indicate that H2O2 activates an ATP-sensitive, Ca2+-independent K+ conductance in LLC-PK1 cells.

Pharmacologically novel GABA receptor in human dorsal root ganglion neurons.

1. Whole cell voltage-clamp studies of gamma-aminobutyric acid (GABA) receptors were performed on large (> 80 microns) cultured human dorsal root ganglion (DRG) neurons. 2. GABA and pentobarbital sodium when applied in micromolar concentrations evoked inward Cl- currents in DRG neurons voltage clamped at negative membrane potentials. 3. Diazepam (10 microM) and pentobarbital (10 microM) upmodulated the GABA current by approximately 149 and 168%, respectively. 4. The GABA currents in human DRG cells were unaffected by the classical GABA antagonists picrotoxin and bicuclline (100 microM). In contrast, the GABA responses evoked in adult rat DRG cells cultured in an identical manner were inhibited by both antagonists. The glycine receptor antagonist strychnine (100 microM) did not alter GABA currents in human DRG cells. 5. Human DRG cells did not respond to glycine (10-100 microM) or taurine (10-100 microM). The GABAB agonist baclofen had no effect on the holding current when patch pipettes were filled with 130 mM KCl. The GABAB antagonists saclofen applied either alone or with GABA was without effect. 6. The differences between the GABA receptors described here and GABA receptors in other species may reflect the presence of receptor subunits unique to human DRG cells.

Inward currents underlying action potentials in rat adrenal chromaffin cells.

1. Current- and voltage-clamp studies were conducted on isolated rat adrenal chromaffin cells to identify the voltage-dependent ion channels mediating inward currents. 2. Mean resting membrane potential of the isolated cells was -62 +/- 3 (SE) mV. Evoked action potentials were both Na+ and Ca2+ based, and whole cell voltage-clamp studies in normal saline revealed an inward-rectifier-type current. 3. Na+ channels were studied in isolation and showed a half-inactivation of -60 +/- 2 mV with a slope factor of -6 mV and a half-activation of -26.8 +/- 2 mV with a slope factor of 6.5 +/- 0.7 mV. 4. Isolated Ca2+ currents, elicited in 10 mM external Ca2+, revealed a T-type current in a subset of cells. Ca2+ currents were sensitive to both N- and L-type channel antagonists, and blockade of the current by the L-type channel antagonist nimodipine and the N-type channel antagonist omega-conotoxin GVIA revealed a third Ca2+-current component that was unaffected by the P-type channel antagonist omega-agatoxin IVA. 5. Ca2+ currents were facilitated 5-20% by a depolarizing prepulse, and facilitation was completely blocked by nimodipine. The effects of the dihydropyridine L-type channel agonist, (+)202-791 and depolarizing prepulses on the currents were additive. 6. The results of this study show that the properties of voltage-dependent ion channels in rat chromaffin cells differ from those reported in their counterparts in bovine chromaffin cells. Na+ channels differ in activation and inactivation properties and Ca2+ channels differ in activation, sensitivity to antagonists, and the magnitude of voltage-dependent facilitation.

Tachykinins activate nonselective cation currents in canine colonic myocytes

The mechanism of tachykinin-induced excitation was studied in isolated colonic muscle cells and intact muscle strips. In whole cell voltage-clamp studies performed at 33 degrees C, neurokinin A (NKA) and substance P (SP) reduced L-type Ca2+ current. NKA and SP activated a cationic current that reversed near 0 mV. This current (INKA or ISP, respectively) had properties similar to the acetylcholine (ACh)-activated nonselective cation conductance (IACh), activated by muscarinic stimulation in other gastrointestinal smooth muscle cells. INKA and ISP were decreased when external Na+ was reduced. In contrast to IACh, INKA and ISP were not facilitated by increases in internal Ca2+, but little or no current was activated by these peptides when extracellular Ca2+ was low. INKA (10(-7) M) and ISP (10(-5) M) were blocked by Cd2+ (5 x 10(-4) M), quinine (10(-3) M), and the tachykinin-receptor antagonist [D-Pro2,D-Trp7,9]SP (10(-5) M). Current clamp recordings and intracellular recordings of intact tissues showed that NKA and SP depolarized the cell membrane, which is consistent with the activation of a nonselective cation conductance. These data suggest that a primary mechanism of the tachykinins is to activate a nonselective cation conductance that leads to depolarization. The increase in Ca2+ entry due to tachykinin stimulation appears to be secondary to the activation of the nonselective cation conductance.

AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity

Cerebellar Purkinje cells are selectively vulnerable to ischemia, although the reasons for this are unknown. In cultured embryonic rat cerebellar neurons, the steady state responses to the desensitizing agonist AMPA relative to responses to the nondesensitizing agonist kainate were greater in Purkinje cells compared to other cells, as measured by whole cell voltage clamp studies. Fluorimetric [Ca2+]i imaging experiments similarly found greater responses to AMPA relative to kainate in Purkinje cells than in other cerebellar neurons. In toxicity experiments measuring cell survival 24 hr following agonist exposure, AMPA and glutamate produced Ca(2+)-dependent toxicity which was selective for the Purkinje cell fraction of the neurons, whereas kainate produced nonselective toxicity, and NMDA selectively spared the mature Purkinje cells. Cyclothiazide, which inhibits AMPA receptor desensitization, enhanced steady state current responses to AMPA and increased the toxicity of AMPA. We conclude that the vulnerability of cerebellar neurons in culture to glutamate agonist-induced toxicity parallels the magnitude of the steady state currents produced, and that Purkinje cells may be selectively vulnerable because they express AMPA receptors which undergo less complete desensitization.

Separation and identification of multiple potassium currents regulating the pacemaker activity of insect neurosecretory cells (DUM neurons)

1. Whole cell voltage-clamp studies performed in isolated adult neurosecretory cells identified as dorsal unpaired median (DUM) neurons of the terminal abdominal ganglion of the cockroach Periplaneta americana have allowed us to reveal a complex voltage-dependent outward current regulating the pacemaker activity. 2. The global outward current remaining after tetrodotoxin treatment was activated by depolarization above -50 mV, showing steep voltage dependence and outward rectification. 3. We used tail current analysis to determine the ionic selectivity of this outward current. The reversal potentials for two extracellular potassium concentrations (-92.7 and -65.4 mV for 3.1 and 10 mM, respectively) is consistent with the expected equilibrium potential for potassium ions. 4. Both peak and sustained components of the global outward K+ current were reduced by external application of 20 mM tetraethylammonium chloride, 10 nM iberiotoxin, 1 nM charybdotoxin (CTX) and 1 mM cadmium chloride. Subtraction of current recorded in CTX solution from that in control solution revealed an unusual biphasic Ca(2+)-dependent K+ current. The fast transient current resistant to 5 mM 4-aminopyridine (4-AP) is distinguished by its dependence on holding potential and time course from the late sustained current. 5. In addition, two other components of CTX-resistant outward K+ current could be separated by sensitivity to 4-AP, time course, and voltage dependence. Beside a calcium-independent delayed outwardly rectifying current, a 4-AP-sensitive fast transient current resembling the A-current has been also identified. It activates at negative potential (about -65 mV) and unlike the A-current of other neurons, it inactivates rapidly with complex inactivation kinetics. A-like current is half-inactivated at -63.5 mV and half-activated at -35.6 mV. 6. Our findings demonstrate for the first time in DUM neuron cell bodies the existence of multiple potassium currents underlying the spontaneous electrical activity. Their identification and characterization represent a fundamental step in further understanding the pacemaker properties of these insect neurosecretory cells.

Inhibitory effects of cocaine on Ca2+ transients and contraction in single cardiomyocytes

The effects of cocaine on the Ca2+ transient responsible for excitation-contraction coupling were studied in single rat heart cells loaded with the fluorescent Ca2+ indicator fura 2. A high-speed imaging technique using a charge-coupled device as detector and transient image store [O'Rourke et al., Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H230-H242, 1990] was used to measure cytosolic free Ca2+ concentration ([Ca2+]i) and contraction simultaneously from the images of fluorescence. Cardiomyocytes maintained a basal [Ca2+]i of approximately 140 nM in presence or absence of cocaine. After electrical field stimulation, [Ca2+]i increased to a peak of 498 +/- 25 nM under control conditions. This was reduced to a peak [Ca2+]i of 389 +/- 25 nM after treatment with 50 microM cocaine. Cocaine also reduced the rate of rise of [Ca2+]i but did not affect the time to peak or the half time for resequestration of the released Ca2+. The rate and extent of cell shortening was reduced by cocaine in parallel with the inhibition of the [Ca2+]i transient. Cocaine had no effect on the half time for relaxation. Cocaine did not modify the relationship between contraction and the elevation of [Ca2+]i over a range of extracellular Ca2+ concentrations. The intracellular pool of Ca2+ releasable by caffeine was also unaffected by cocaine. In the presence of the beta-adrenergic agonist isoproterenol, which caused a large enhancement of peak [Ca2+]i and contraction, cocaine still inhibited both parameters. However, cocaine did not reverse the ability of isoproterenol to enhance the rate of Ca2+ reuptake and cell relaxation. Whole cell voltage-clamp studies showed that 50 microM cocaine reduced both the Na+ current (50%) and the Ca2+ current (30%). These data suggest that sarcolemmal ion channels are the primary site that, in cardiac muscle, mediate the negative inotropic effects and the suppression of [Ca2+]i transients by cocaine.

Role of nonselective cation current in muscarinic responses of canine colonic muscle.

The mechanism of muscarinic excitation was studied in colonic muscle strips and isolated cells. In whole cell voltage-clamp studies performed at 33 degrees C utilizing the permeabilized patch technique, acetylcholine (ACh) reduced an L-type Ca2+ current. With K+ currents blocked, depolarization to positive potentials in the presence of ACh elicited outward current. Difference currents showed that ACh activated a voltage-dependent current that reversed at about -8 mV; this current (IACh) had properties similar to the nonselective cation conductance found in other smooth muscle cells. The reversal potential of IACh shifted toward negative potentials when external Na+ was reduced, and the inward current elicited at -70 mV decreased when external Na+ was reduced. IACh was facilitated by internal Ca2+. After the current was activated at a holding potential of -70 mV, depolarizations to -30 to 0 mV elicited influx of Ca2+ via voltage-dependent Ca2+ channels. After repolarization to the holding potential, a large inward tail current was observed. IACh was blocked by Ni2+ and Cd2+ at concentrations of 100 microM or less. Quinine (0.5 mM) also blocked IACh. With the use of the sensitivity of IACh to reduced external Na+ and divalent cations, the role of IACh in responses of intact muscles to ACh was examined. When external Na+ was reduced, ACh failed to increase slow-wave duration, and Ni2+ (50 microM) reversed the depolarization caused by ACh. These data suggest an important role for IACh in the electrical responses of colonic muscles. The contribution of IACh appears to prolong slow waves, which would allow greater entry of Ca2+ and increased force development.

Isolated supporting cells from the organ of Corti: Some whole cell electrical characteristics and estimates of gap junctional conductance

Whole cell voltage clamp studies were performed upon isolated and small groups of supporting cells from the guinea pig organ of Corti in order to evaluate junctional and non-junctional membrane characteristics. Single Hensen cells have an average input resistance and capacitance of 1.03 GΩ and 24.9 pF, respectively. I–V functions indicate an outward K + rectification, which is blocked by external TEA, intracellular Cs, or photo-irradiation of intracellularly injected fluorescent dye. Voltage clamping of pairs or small groups of cells indicates that supporting cells ‘share’ K + channels within the syncitium. The input impedance of coupled cells was studied during uncoupling with CO 2 or octanol media. As expected, coupled cells showed an increase in input capacitance and a decrease in input resistance over single cell values. Input capacitance is a more sensitive indicator of cell coupling than dc input resistance. During uncoupling, input capacitance values drop to single cell levels prior to an increase of dc input resistance to single cell levels. Modeling the results indicates that Hensen cells are well-coupled under normal conditions and may have junctional resistances with values less than 0.1% of the non-junctional resistance. The sensitivity of the supporting cell syncitium's input impedance to small changes in junctional resistance markedly influences the syncitium's RC filter characteristics, and thus may control the frequency response of sound evoked electrical activity measurable in supporting cells in vivo.