Peak Outward Current Research Articles

The residence of synaptically released dopamine on D2 autoreceptors.

SUMMARYNeuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition.

Reduced Excitability and Increased Neurite Complexity of Cortical Interneurons in a Familial Mouse Model of Amyotrophic Lateral Sclerosis.

Cortical interneurons play a crucial role in regulating inhibitory-excitatory balance in brain circuits, filtering synaptic information and dictating the activity of pyramidal cells through the release of GABA. In the fatal motor neuron (MN) disease, amyotrophic lateral sclerosis (ALS), an imbalance between excitation and inhibition is an early event in the motor cortex, preceding the development of overt clinical symptoms. Patients with both sporadic and familial forms of the disease exhibit reduced cortical inhibition, including patients with mutations in the copper/zinc superoxide-dismutase-1 (SOD1) gene. In this study, we investigated the influence of the familial disease-causing hSOD1-G93A ALS mutation on cortical interneurons in neuronal networks. We performed whole-cell patch-clamp recordings and neurobiotin tracing from GFP positive interneurons in primary cortical cultures derived from Gad67-GFP::hSOD1G93A mouse embryos. Targeted recordings revealed no overt differences in the passive properties of Gad67-GFP::hSOD1G93A interneurons, however the peak outward current was significantly diminished and cells were less excitable compared to Gad67-GFP::WT controls. Post hoc neurite reconstruction identified a significantly increased morphological complexity of the Gad67-GFP::hSOD1G93A interneuron neurite arbor compared to Gad67-GFP::WT controls. Our results from the SOD1 model suggest that cortical interneurons have electrophysiological and morphological alterations that could contribute to attenuated inhibitory function in the disease. Determining if these phenomena are driven by the network or represent intrinsic alteration of the interneuron may help explain the emergence of inhibitory susceptibility and ultimately disrupted excitability, in ALS.

Calcium- and ATP-dependent regulation of Na/Ca exchange function in BHK cells: Comparison of NCX1 and NCX3 exchangers

Na+/Ca2+ exchangers (NCX) mediate bidirectional Ca2+ fluxes across cell membranes and contribute to Ca2+ homeostasis in many cell types. Exchangers are regulated by gating reactions that depend on Na+ and Ca2+ binding to transport and regulatory sites. A Na+i-dependent inactivation is prominent in all isoforms, whereas Ca2+i-dependent regulation varies among isoforms. Here we characterize new details of NCX operation and describe differences and similarities between NCX3 and NCX1 regulation by intracellular Ca2+ and ATP. To compare isoforms, we employed BHK cells expressing NCX3 or NCX1 constitutively and exchange activity was analysed in whole-cell and excised patch recordings under “zero-trans” conditions (i.e., with only one transported ion species on each side). Using BHK cells with low cytoplasmic Ca2+ buffering, outward (reverse) currents, reflecting Ca2+ influx, are activated by applying extracellular Ca2+ (Cao) in the presence of Na+ on the cytoplasmic side. When firstly activated, peak outward NCX3 currents rapidly decay over seconds and then typically develop a secondary transient peak with slower kinetics, until Cao removal abolishes all outward current. The delayed rise of outward current is the signature of an activating process since peak outward NCX3 currents elicited at subsequent Cao bouts remain stimulated for minutes and slower decline towards a non-zero level during continued Cao application. Secondary transient peaks and current stimulation are suppressed by increasing the intracellular Ca2+ buffer capacity or by replacing cytoplasmic ATP with the analogues AMP-PNP or ATPγS. In BHK cells expressing NCX1, outward currents activated under identical settings decay to a steady-state level during single Cao application and are significantly larger, causing strong and long-lived run down of subsequent outward currents. NCX1 current run down is not prevented by increasing cytoplasmic Ca2+ buffering but secondary transient peaks in the outward current profile can be resolved in the presence of ATP. Finally, inward currents recorded in patches excised from NCX3-expressing cells reveal a proteolysis-sensitive, Ca-dependent inactivation process that is unusual for NCX1 forward activity. Together, our results suggest that NCX function is regulated more richly than appreciated heretofore, possibly including processes that are lost in excised membrane patches.

Ionic Basis of Repolarization of Atrial and Ventricular Specific Cell Types Derived from Human Induced Pluripotent Stem Cells

Cardiomyocytes derived from human induced pluripotent stem cells (hIPSC-CMs) are an innovative cellular system for understanding human cardiac pathology and physiology. However, hIPSC-CMs express low levels of inward rectifying potassium channel (IK1) relative to native cardiac cells. The lack of this current deforms the action potential (AP) and leads to a depolarized or spontaneous diastolic potential. We used electronic expression of IK1 via dynamic clamp to restore the resting membrane potential back to physiological levels. This resulted in an improved AP morphology, including a reduction in variability and a rate dependent spike and dome shape. There were several significant differences between atrial and ventricular cells including differences in cell capacitance, sodium current magnitude and kinetics and most prominently differences in the transient outward currents. The late components of outward current, cells with atrial APs had a significantly larger sustained outward IKUR or Kv1.5-like component at +50 mV than ventricular shaped APs (in pA/pF: 3.71 ± 0.55 (n=5) vs 1.00 ± 0.10 (n=16),P<0.05) but similar peak outward currents: (6.89 ± 0.50 (n=5) vs. 6.58 ± 0 .67 (n=14),P=N.S). This plateau current is strongly inhibited by 50 micro molar 4-aminopyridine(4-AP). Similarly, atrial-like APs took on a ventricular like shape when treated with 4-AP while the ventricular myocytes APs were 4-AP insensitive. A cloned Kv1.5 current was expressed in oocytes and was used through the electronic expression system to add an IKur to the ventricular myocytes. Addition of this current changed the action potential morphology from ventricle to atrial like. This strongly suggests that IKur is the major determinant of atrial action potential morphology.

A Comparison of Acutely Isolated Human Ventricular Myocytes with Stem Cell Derived Cardiocytes

Recent studies have characterized cardiac ionic currents in cardiocytes derived from human induced pluripotent stem cells. However, direct comparison with human ventricular myocytes is hampered by differences in experimental conditions. Similarly, stem cell derived cardiocytes are different and potentially more variable than native myocytes. We examined commercially prepared cells (iCells) and scale production cells (hIPS-CMs) using electronic expression of IK1 to distinguish ventricular cells, compared with myocytes (VM) from biopsy samples.APs were recorded in VM, iCells and hiPSC-CMs. iCells and hiPSC-CMs had depolarized membrane potentials, slower upstroke velocities, and prolonged AP relative to VM. These differences were minimized by electronic expression of IK1. Capacitances were larger in VM (298.97 ± 27.5 pF (4 hearts, n=44)) than in iCells (73.21 ± 5.4 (n=32), p<0.05) and hIPS-CMs (64.25 ± 4.94 (n=75), p<0.05). At −120 mV, a large IK1 was observed in VM (−14.3 ± 1.36 pA/pF (4 hearts, n=44)); IK1 was much smaller in iCells (−1.73 ± 0.92 (n=19), p<0.01) and hIPS-CMs (−2.54 ± 0.49 (n=29), p<0.01). VM had larger peak outward (Ito) currents at +50 mV (6.2 ±- 0.7 pA/pF (n=44, four hearts)) than iCells (2.6 ± 0.2 (n=19, p<0.01)) and hIPS-CMs (2.9 ± 0.3 (n=29, p<0.01)) but all had similar variability relative to current density. Large sodium currents were recorded at 0 mV in VM (−23.7 +/− 3.4 pA/pF (4 hearts, 32 myocytes)) as well as in iCells (−26.1 +/− 3.2 (n=14)) and in hiPSC-CMs (−17.0 +/− 4.1 (n=16)) with high degrees of variability for all cell types. In conclusion, there are functional differences between VM and stem cell derived cell types, but minimal differences between stem cell derived cardiocytes based on source. Similarly, cell to cell variability was similar regardless of source.

Human Electrophysiological and Pharmacological Properties of XEN-D0101

The human electrophysiological and pharmacological properties of XEN-D0101 were evaluated to assess its usefulness for treating atrial fibrillation (AF). XEN-D0101 inhibited Kv1.5 with an IC50 of 241 nM and is selective over non-target cardiac ion channels (IC50 Kv4.3, 4.2 μM; hERG, 13 μM; activated Nav1.5, >100 μM; inactivated Nav1.5, 34 μM; Kir3.1/3.4, 17 μM; Kir2.1, >>100 μM). In atrial myocytes from patients in sinus rhythm (SR) and chronic AF, XEN-D0101 inhibited non-inactivating outward currents (Ilate) with IC50 of 410 and 280 nM, respectively, and peak outward currents (Ipeak) with IC50 of 806 and 240 nM, respectively. Whereas Ilate is mainly composed of IKur, Ipeak consists of IKur and Ito. Therefore, the effects on Ito alone were estimated from a double-pulse protocol where IKur was inactivated (3.5 µM IC50 in SR and 1 µM in AF). Thus, inhibition of Ipeak is because of IKur reduction and not Ito. XEN-D0101 significantly prolonged the atrial action potential duration at 20%, 50%, and 90% of repolarization (AF tissue only) and significantly elevated the atrial action potential plateau phase and increased contractility (SR and AF tissues) while having no effect on human ventricular action potentials. In healthy volunteers, XEN-D0101 did not significantly increase baseline- and placebo-adjusted QTc up to a maximum oral dose of 300 mg. XEN-D0101 is a Kv1.5/IKur inhibitor with an attractive atrial-selective profile.

Quantification of Transmembrane Currents during Action Potential Propagation in the Heart

The measurement, quantitative analysis, theory, and mathematical modeling of transmembrane potential and currents have been an integral part of the field of electrophysiology since its inception. Biophysical modeling of action potential propagation begins with detailed ionic current models for a patch of membrane within a distributed cable model. Voltage-clamp techniques have revolutionized clinical electrophysiology via the characterization of the transmembrane current gating variables; however, this kinetic information alone is insufficient to accurately represent propagation. Other factors, including channel density, membrane area, surface/volume ratio, axial conductivities, etc., are also crucial determinants of transmembrane currents in multicellular tissue but are extremely difficult to measure. Here, we provide, to our knowledge, a novel analytical approach to compute transmembrane currents directly from experimental data, which involves high-temporal (200 kHz) recordings of intra- and extracellular potential with glass microelectrodes from the epicardial surface of isolated rabbit hearts during propagation. We show for the first time, to our knowledge, that during stable planar propagation the biphasic total transmembrane current (Im) dipole density during depolarization was ∼0.25 ms in duration and asymmetric in amplitude (peak outward current was ∼95 μA/cm2 and peak inward current was ∼140 μA/cm2), and the peak inward ionic current (Iion) during depolarization was ∼260 μA/cm2 with duration of ∼1.0 ms. Simulations of stable propagation using the ionic current versus transmembrane potential relationship fit from the experimental data reproduced these values better than traditional ionic models. During ventricular fibrillation, peak Im was decreased by 50% and peak Iion was decreased by 70%. Our results provide, to our knowledge, novel quantitative information that complements voltage- and patch-clamp data.

μ-Calpain-mediated deregulation of cardiac, brain, and kidney NCX1 splice variants

μ-Calpain is a Ca2+-activated protease abundant in mammalian tissues. Here, we examined the effects of μ-calpain on three alternatively spliced variants of NCX1 using the giant, excised patch technique. Membrane patches from Xenopus oocytes expressing either heart (NCX1.1), kidney (NCX1.3), or brain (NCX1.4) variants of NCX1 were exposed to μ-calpain and their Na+-dependent (I1) and Ca2+-dependent (I2) regulatory phenotypes were assessed. For these exchangers, I1 inactivation is evident as a Na+i-dependent decay of peak outward currents whereas I2 regulation manifests as outward current activation by micromolar Ca2+i concentrations. Notably, with NCX1.1 and NCX1.4 but not in NCX1.3, higher Ca2+i levels alleviate I1 inactivation. Our results show that (i) μ-calpain selectively ablates Ca2+-dependent (I2) regulation leading to a constitutive activation of exchange current, (ii) μ-calpain has much smaller effects on Na+-dependent (I1) regulation, produced by a slight destabilization of the I1 state, and (iii) Ca2+-dependent regulation (I2) and Ca2+-mediated alleviation of I1 appear to be functionally distinct mechanisms, the latter of which is left largely intact after μ-calpain treatment. The ability of μ-calpain to selectively and constitutively activate Na+–Ca2+ exchange currents may have important pathophysiological implications in tissue where these splice variants are expressed.

The Effects of Ginsenoside Rg3 on Human Kv1.4 Channel Currents without the N-Terminal Rapid Inactivation Domain

Kv1.4 channel belongs to the family of voltage-gated potassium channels that mediate transient and rapidly inactivating A-type currents and N-type inactivation. This N-type inactivation can be removed by the deletion of N-terminal domains, which exhibit non-inactivating currents and C-type inactivation. In our previous report, we demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active ingredients of ginseng saponins, inhibits human Kv1.4 (hKv1.4) channel currents through the interaction with amino acids, including Lys (K) residue, which is known as K(+) activation and the extracellular tetraethylammonium (TEA) binding site. In the present study, we examined the effects of Rg(3) on hKv1.4 channel currents without the N-terminal rapid inactivation domain. We constructed hKv1.4Delta2-61 channels by N-terminal deletion of 2-61 amino acid residues. We investigated the effect of Rg(3) on hKv1.4Delta2-61 channel currents. We found that Rg(3) preferentially inhibited non-inactivating outward currents rather than peak outward currents of hKv1.4Delta2-61 channels. The mutation of K531 hKv1.4Delta2-61 to K531Y hKv1.4Delta2-61 and raising of extracellular [K(+)](o) abolished Rg(3) inhibitions on non-inactivating outward currents. Rg(3) treatment increased the C-type inactivation rate, but raising the extracellular [K(+)](o) reversed Rg(3) action. These results provide additional evidence that K531 residue also plays an important role in the Rg(3)-mediated non-inactivating current blockages and in Rg(3)-mediated increase of the C-type inactivation rate in hKv1.4Delta2-61 channels.

Association of Kv1.5 and Kv1.3 Contributes to the Major Voltage-dependent K+ Channel in Macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.

Activation of Potassium Channels by Tempol in Arterial Smooth Muscle Cells From Normotensive and Deoxycorticosterone Acetate-Salt Hypertensive Rats

Large-conductance Ca(2+)-activated potassium (BK) channels modulate vascular tone. Tempol, an O(2)(-) dismutase mimetic, causes vasodilation via activation of vascular BK channels. In this study, we investigated the mechanisms underlying tempol-induced activation of BK channels in mesenteric arterial (MA) myocytes from sham and deoxycorticosterone acetate (DOCA)-salt hypertensive rats. In sham myocytes, whole-cell patch clamp studies showed that tempol enhanced peak outward currents (I(o)). This effect was larger in DOCA-salt myocytes. Tempol caused a leftward shift in the activation curve for I(o) in sham and DOCA-salt myocytes. In DOCA-salt myocytes, the peak I(o) at +80 mV did not differ from sham myocytes, but iberiotoxin (BK channel blocker) caused a larger reduction of I(o) in DOCA-salt compared with sham myocytes. Iberiotoxin but not 4-aminopyridine blocked the I(o) activated by tempol. Tiron, another O(2)(-) scavenger, had no effect on I(o). Using inside-out patches, we found that tempol caused a 4-fold increase in open probability (P(o)) of BK channels but did not change the mean channel open time in sham and DOCA-salt myocytes. Tempol did not change single channel conductance in sham or DOCA-salt myocytes. Western blot and immunocytochemical studies revealed that BK channel alpha-subunit expression was increased in DOCA-salt MA compared with sham MA. The data indicate that tempol directly activates BK channels by increasing channel P(o). We conclude that upregulation of the BK channel alpha-subunit protein and tempol-induced increases in BK channel P(o) contribute to the enhanced depressor response caused by tempol in DOCA-salt hypertensive rats.

The Synaptic Mechanism of Direction Selectivity in Distal Processes of Starburst Amacrine Cells

Patch-clamp recordings revealed that distal processes of starburst amacrine cells (SACs) received largely excitatory synaptic input from the receptive field center and nearly purely inhibitory inputs from the surround during both stationary and moving light stimulations. The direct surround inhibition was mediated mainly by reciprocal GABA(A) synapses between opposing SACs, which provided leading and prolonged inhibition during centripetal stimulus motion. Simultaneous Ca(2+) imaging and current-clamp recording during apparent-motion stimulation further demonstrated the contributions of both centrifugal excitation and GABA(A/C)-receptor-mediated centripetal inhibition to the direction-selective Ca(2+) responses in SAC distal processes. Thus, by placing GABA release sites in electrotonically semi-isolated distal processes and endowing these sites with reciprocal GABA(A) synapses, SACs use a radial-symmetric center-surround receptive field structure to build a polar-asymmetric circuitry. This circuitry may integrate at least three levels of interactions--center excitation, surround inhibition, and reciprocal inhibitions that amplify the center--surround antagonism-to generate robust direction selectivity in the distal processes.

Modulation of A-type potassium currents in retinal horizontal cells by extracellular calcium and zinc

Extracellular Ca2+ and Zn2+ influence many aspects of retinal function. Here, we examined the effect of external Ca2+ and Zn2+ on potassium channels of retinal horizontal cells. When extracellular Ca2+ was lowered from 3 mM to 0.3 mM, horizontal cell transient outward currents elicited by voltage steps from resting membrane potential (-70 mV) were decreased by approximately 50%, whereas the sustained currents remained unchanged. This effect was due to a hyperpolarizing shift in the steady-state inactivation curve of A-type K+ currents when extracellular Ca2+ concentration was lowered. The mean half inactivation potential of the steady-state inactivation curves was hyperpolarized from -56.3 +/- 4.7 mV in 3 mM Ca2+ to -76.4 +/- 3.9 mV in 0.3 mM Ca2+. Neither the state-steady activation curve nor the kinetics of inactivation was significantly changed in low extracellular Ca2+. The addition of 30 microM Zn2+ restored peak outward currents in 0.3 mM Ca2+. The half inactivation voltages were depolarized from -70 +/- 2.8 mV in 0.3 mM Ca2+ to -56 +/- 2.6 mV in 0.3 mM Ca2+ plus 30 microM Zn2+. Taken together, the results indicate that external Ca2+ and Zn2+ maintain the activity of A-type potassium channels in retinal horizontal cells by influencing the voltage dependence of steady-state inactivation.

The relation between potassium channel properties and proliferation of human lung adenocarcinoma cells

Oncogenesis, development, invasion and metastasis of lung cancer are modulated by relative genes of lung cancer, and the expression, deletion or mutation of these genes are regulated by cell membrane signal transduction system and cell membrane ionic channels. The aim of this study is to explore the electrophysiological properties of human lung adenocarcinoma cell line A549 and cell proliferation affected by tetraethylammonium chloride (TEA), one potassium channel blocker, so as to know whether the voltage-gated potassium channels are required for the proliferation of A549 cell line. Ionic currents were recorded by whole-cell patch-clamp recording technique. The proliferation activity of A549 cells was measured by MTT assay. Membrane current was observed when cells were held at -70mV and test potentials ranged from -30 to +110mV. The current exhibited properties of voltage-dependent, outward rectification and no or little inactivation over the 500ms voltage pulse. Exposure of tumor cells to 10mmol/L TEA reduced the peak outward potassium current (evoked by depolarization to +110mV) from (1057.52±59.17)pA to (212.26±11.96)pA, the ratio of suppression was 79.92% (P < 0.01). Obvious inhibitive effect of TEA with different concentrations ranged from 20 to 60mmol/L on proliferation activity of the cells was found. The voltage-gated potassium channels exist in human lung adenocarcinoma cell line A549 and play a great role in proliferation of A549 cells. TEA can inhibit proliferation of A549 cell through blocking the potassium channels and the inhibition is dose-dependent.

CDNA Cloning and Expression of the Cardiac Na+/Ca2+ Exchanger from Mozambique Tilapia (Oreochromis mossambicus) Reveal a Teleost Membrane Transporter with Mammalian Temperature Dependence

The complete cDNA sequence of the tilapia cardiac Na(+)/Ca2+ exchanger (NCX-TL1.0) was determined. The 3.1-kb transcript encodes a protein 957 amino acids in length, with a predicted signal peptide cleaved at residue 31 and two potential N-glycosylation sites in the extracellular N terminus. Hydropathy analysis and sequence comparison predicted a mature protein with nine transmembrane-spanning segments, consistent with the structural topologies of other known mammalian and teleost NCX isoforms. Overall sequence comparison shows high identity to both trout NCX-TR1.0 ( approximately 81%) and mammalian NCX1.1 ( approximately 73%), and phylogenetic analyses confirmed its identity as a member of the NCX1 gene family, expressing exons A, C, D, and F in the alternative splice site. Sequence identity is even higher in the alpha-repeats, the exchanger inhibitory peptide (XIP) site, and Ca(2+)-binding domains, which is reflected in the functional and regulatory properties of tilapia NCX-TL1.0. When NCX-TL1.0 was expressed in Xenopus oocytes and the currents were measured in giant excised patches, they displayed both positive regulation by Ca2+ and Na(+)-dependent inactivation in a manner similar to trout NCX-TR1.0. However, tilapia NCX-TL1.0 exhibited a relatively high sensitivity to temperature compared with trout NCX-TR1.0. Whereas trout NCX-TR1.0 currents displayed activation energies of approximately 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 +/- 9 and 67 +/- 21 kJ/mol, respectively. Using comparative sequence analysis, we highlighted 10 residue positions in the N-terminal domain of the NCX that, in combination, may confer exchanger temperature dependence through subtle changes in protein flexibility. Tilapia NCX-TL1.0 represents the first non-mammalian NCX to exhibit a mammalian temperature dependence phenotype and will prove to be a useful model in defining the interplay between molecular flexibility and stability in NCX function.

Actions of the anti-oestrogen agent clomiphene on outward K+ currents in rat ventricular myocytes.

1. The effects of clomiphene (CLM) on cardiac outward K+ current components from rat isolated ventricular myocytes were investigated using the whole-cell patch-clamp technique. Clomiphene (10 micromol/L) significantly inhibited both peak (Ipeak) and end-pulse (Ilate) outward currents (elicited by a 500 msec voltage step from -40 to +50 mV in the presence of K+-containing intracellular and extracellular solutions) by approximately 37% (n = 6; P < 0.01) and 49% (n = 6; P < 0.01), respectively. In contrast, CLM had no effect on outward currents when K+-free solutions were used. 2. A double-pulse protocol and Boltzmann fitting were used to separate individual K+ current components on the basis of their voltage-dependent inactivation properties. At potentials positive to -80 mV, two inactivating transient outward components (Ito) and (IKx) and a non-inactivating steady state component (Iss) could be distinguished. 3. Clomiphene inhibited both Ito and Iss. The maximal block of Ito and Iss induced by CLM (100 micromol/L) was approximately 61% (n = 5) and 43% (n = 5) with IC50 values of 1.54 +/- 0.39 and 2.2 +/- 0.4 micromol/L, respectively. In contrast, the peak magnitude of IKx was unaltered by CLM, although its time-course of inactivation was accelerated. 4. Further experiments whereby myocytes were superfused with the vasoactive peptide endothelin (ET)-1 (20 nmol/L) revealed that CLM (10 micro mol/L) completely abolished the ET-1-sensitive component of Iss. 5. Our findings demonstrate, for the first time, the effects of CLM on distinct cardiac K+ current components and show that CLM modulates the voltage-gated K+ current components Ito and IKx and inhibits the steady state outward current Iss in rat ventricular myocytes.

Partial hippocampal kindling increases GABA B receptor-mediated postsynaptic currents in CA1 pyramidal cells

In previous studies, we showed that partial hippocampal kindling decreased the efficacy of the presynaptic GABA B receptors on both GABAergic and glutamatergic terminals of CA1 neurons in hippocampal slices in vitro. In this study, GABA B receptor-mediated inhibitory postsynaptic currents (GABA B-IPSCs) were assessed by whole-cell recordings in CA1 pyramidal neurons in hippocampal slices of male Long–Evans rats. The peak GABA B-IPSC evoked by a brief train of supramaximal stratum radiatum stimuli (20 pulses of 300 Hz) in the presence of picrotoxin (0.1 mM) and kynurenic acid (1 mM) was larger in neurons of kindled (65.9±5.2 pA, N=42 cells) than control (45.8±4.8 pA, N=32 cells) rats ( P<0.01). Adding GABA uptake blocker nipecotic acid (1 mM) or GABA B receptor agonist baclofen (0.01 mM) in the perfusate induced outward currents that were blocked by GABA B receptor antagonist CGP 55845A (1 μM). The peak outward current induced by nipecotic acid was larger in neurons of the kindled (55.4±5.7 pA, N=30) than the control group (39.8±4.5 pA, N=28) ( P<0.05). However, the magnitude of the baclofen-induced current was not different between kindled (90.8±6.9 pA, N=29) and control (87.2±5.9 pA, N=21) groups ( P>0.05). We concluded that partial hippocampal kindling increased GABA B-IPSCs in hippocampal CA1 pyramidal cells via multiple presynaptic mechanisms.

Protein kinase A hyperphosphorylation increases basal current but decreases beta-adrenergic responsiveness of the sarcolemmal Na+-Ca2+ exchanger in failing pig myocytes.

The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by beta-adrenergic receptor (beta-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P<0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P<0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P<0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to beta-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominantly attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although beta-AR responsiveness is decreased because of NCX hyperphosphorylation.

Effects of dihydrotestosterone on cardiac inward rectifier K(+) current.

There are well-described sexually dimorphic differences both in the electrocardiogram and in the propensity to develop drug-induced arrhythmias. The QT interval and the risk of ventricular proarrhythmia are reduced in males compared with females. Inward rectifier potassium current (IK(1)) is a primary determinant of the ventricular resting membrane potential, and an important contributor to myocardial excitability. Using the whole-cell patch-clamp technique, we evaluated the effects of dihydrotestosterone (DHT) on IK(1) in ventricular myocytes from castrated rabbits that were treated with either replacement DHT or vehicle-control for 3 weeks. Compared with the DHT-treated group, myocytes from the control animals had a significant reduction in inward IK(1) conductance (p < 0.005) and rectification ratio (RR) (p < 0.04) with no significant change in peak outward current. Acute DHT superfusion of the myocytes increased inward IK(1) conductance from baseline (p < 0.05) and increased the RR (p < 0.05). Testosterone has been reported to increase intracellular ornithine decarboxylase activity in ventricular tissue, which would increase intracellular polyamines, known modulators of IK(1) rectification. We found that inclusion of the intracellular polyamines spermidine and putrescine in the pipette solution caused a decrease in inward IK(1), accompanied by an increase in peak outward current and a reduction in the RR. In summary, DHT modulates IK(1) in a chronic, as well as, an acute fashion. These effects are not because of altered intracellular polyamines. DHT may modulate myocardial excitability through effects on IK(1).

Voltage dependence of slow inactivation in Shaker potassium channels results from changes in relative K(+) and Na(+) permeabilities.

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.