Cannabinoids and dopamine receptors' action on calcium current in rat neurons.

OBJECTIVE: To study the effects of cannabinoid, glutamate, and dopamine agonists and antagonists on the calcium current rat sympathetic neurons. METHODS: Calcium current was recorded using the whole-cell variant of the patch-clamp technique. After expression in neuronal membranes of the cannabinoid CB1, glutamate mGluR2, or dopamine D1 receptor (by microinjection of the levant receptor's cDNA into the neuron's nucleus) agonists' and antagonists' effects were observed. RESULTS: Applications of agonists of the expressed receptor (0.1-10 microM) decreased the calcium current. The calcium current was increased after application of cannabinoid antagonists (AM251 and AM630); these compounds thus act as inverse agonists in this preparation. Glutamate and dopamine antagonists had no effects on the calcium current by themselves. Combined application of cannabinoids and dopamine, but not glutamate, agonists produced a decrement in the calcium current that was bigger than either of the effects seen when one agonist was applied alone. CONCLUSIONS: These results suggest that cannabinoid with dopamine receptors have an interactive inhibitory effect on the calcium current in this preparation, indicating that within the nervous system, receptor interactions may be important in the regulation of ion-channel functions.

Blockade of currents by the antimalarial drug chloroquine in feline ventricular myocytes.

The effects of the antimalarial drug chloroquine on cardiac action potential and membrane currents were studied at clinically relevant concentrations. In cat Purkinje fibers, chloroquine at concentrations of 0.3 to 10 microM increased action potential duration, and reduced maximum upstroke velocity. At concentrations of 3 and 10 microM, chloroquine increased automaticity and reduced maximum diastolic potential, and after 60 min of perfusion with a concentration 10 microM, spontaneous activity was abolished. In isolated cat ventricular myocytes, chloroquine also increased action potential duration in a concentration-dependent manner, and reduced resting membrane potential at 3 and 10 microM. In voltage-clamped cat ventricular myocytes, chloroquine blocked several inward and outward membrane currents. The order of potency was inward rectifying potassium current (I(K1)) > rapid delayed rectifying potassium current (I(Kr)) > sodium current (I(Na)) > L-type calcium current (I(Ca-L)). Only tonic block of I(Na) and I(Ca-L) was observed at a stimulation frequency of 0.1 Hz and no additional blockade was observed during stimulation trains applied at 1 Hz. The effect of chloroquine on I(K1) was voltage-dependent, with less pronounced blockade at negative test potentials. In addition, unblock was achieved by hyperpolarizing pulses to potentials negative to the current reversal potential. Chloroquine blocked the rapid component of the delayed rectifying outward current, I(Kr,) but not the slow component, I(Ks). These findings provide the cellular mechanisms for the prolonged QT interval, impaired ventricular conduction, and increased automaticity induced by chloroquine, which have been suggested as responsible for the proarrhythmic effects of the drug.

Effects of a quaternary bupivacaine derivative on delayed rectifier K(+) currents.

Block of hKv1.5 channels by R-bupivacaine has been attributed to the interaction of the charged form of the drug with an intracellular receptor. However, bupivacaine is present as a mixture of neutral and charged forms both extra- and intracellularly. We have studied the effects produced by the R(+) enantiomer of a quaternary bupivacaine derivative, N-methyl-bupivacaine, (RB(+)1C) on hKv1.5 channels stably expressed in Ltk(-) cells using the whole-cell configuration of the patch-clamp technique. When applied from the intracellular side of the membrane, RB(+)1C induced a time- and voltage-dependent block similar to that induced by R-bupivacaine. External application of 50 microM RB(+)1C reduced the current at +60 mV by 24+/-2% (n=10), but this block displayed neither time- nor voltage-dependence. External RB(+)1C partially relieved block induced by R-bupivacaine (61+/-2% vs 56+/-3%, n=4, P<0.05), but it did not relieve block induced by internal RB(+)1C. In addition, it did not induce use-dependent block, but when applied in combination with internal RB(+)1C a use-dependent block that increased with pulse duration was observed. These results indicate that RB(+)1C induces different effects on hKv1.5 channels when applied from the intra or the extracellular side of the membrane, suggesting that the actions of bupivacaine are the resulting of those induced on the external and the internal side of hKv1.5 channels.

Differential targeting of Shaker-like potassium channels to lipid rafts.

Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitric-oxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltage-gated K(+) channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K(+) channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.

Phosphorylation is required for alteration of kv1.5 K(+) channel function by the Kvbeta1.3 subunit.

The Kv1.5 K(+) channel is functionally altered by coassembly with the Kvbeta1.3 subunit, which induces fast inactivation and a hyperpolarizing shift in the activation curve. Here we examine kinase regulation of Kv1.5/Kvbeta1.3 interaction after coexpression in human embryonic kidney 293 cells. The protein kinase C inhibitor calphostin C (3 microM) removed the fast inactivation (66 +/- 1.9 versus 11 +/- 0.25%, steady state/peak current) and the beta-induced hyperpolarizing voltage shift in the activation midpoint (V(1/2)) (-21.9 +/- 1.4 versus -4.3 +/- 2.0 mV). Calphostin C had no effect on Kv1.5 alone with respect to inactivation kinetics and V(1/2). Okadaic acid, but not the inactive derivative, blunted both calphostin C effects (V(1/2) = -17.6 +/- 2.2 mV, 38 +/- 1.8% inactivation), consistent with dephosphorylation being required for calphostin C action. Calphostin C also removed the fast inactivation (57 +/- 2.6 versus 16 +/- 0.6%) and the shift in V(1/2) (-22.1 +/- 1.4 versus -2.1 +/- 2.0 mV) conferred onto Kv1.5 by the Kvbeta1.2 subunit, which shares only C terminus sequence identity with Kvbeta1. 3. In contrast, modulation of Kv1.5 by the Kvbeta2.1 subunit was unaffected by calphostin C. These data suggest that Kvbeta1.2 and Kvbeta1.3 subunit modification of Kv1.5 inactivation and voltage sensitivity require phosphorylation by protein kinase C or a related kinase.

Frequency-dependent effects of 4-aminopyridine and almokalant on action-potential duration of adult and neonatal rabbit ventricular muscle.

The effects of 4-aminopyridine (1 mM) and almokalant (1 microM) on action-potential duration of neonatal and adult rabbit ventricular multicellular preparations and plateau membrane currents of single ventricular myocytes were studied. In adult ventricular preparations, 4-aminopyridine increased action-potential duration in a frequency-dependent manner, with a greater effect at low stimulation frequencies ("reverse" use dependence). In neonatal preparations, the increase in action-potential duration by 4-aminopyridine was significantly smaller than in adults, and the effect was frequency independent. Almokalant increased the action-potential duration more in neonatal than in adult myocytes. The effect of almokalant was frequency independent between 0.5 and 2 Hz. The block of transient outward current and delayed rectifier current in single myocytes was quantitatively similar. We propose that differences in the kinetic behavior of the transient outward current between adult and neonatal ventricular preparations, slower inactivation, and recovery from inactivation in adults determine differences in the frequency-dependent changes induced by 4-aminopyridine and almokalant on action-potential duration.

4-aminopyridine activates potassium currents by activation of a muscarinic receptor in feline atrial myocytes.

1. The effects of 4-aminopyridine (4-AP) on action potentials, macroscopic membrane currents and single-channel recording from cardiac left atrial myocytes of the adult cat were studied using the whole-cell and cell-attached configurations of the patch-clamp technique. 2. 4-AP (1 mM) produced a hyperpolarization of the resting membrane potential and a shortening of action potential duration. Under voltage-clamp conditions, we have found that 4-AP increased a background current and a delayed rectifier outward current. These effects were antagonized by atropine. In addition, both effects seemed to be mediated through a pertussis toxin-sensitive G protein. 3. The background current induced by 4-AP displayed properties that are highly similar to those of the inwardly rectifying potassium current activated by acetylcholine (IK(ACh)). The time-dependent potassium current activated by 4-AP has kinetic and pharmacological properties different from those of the delayed rectifier potassium current previously identified in cardiac myocytes. 4. The activation of the delayed rectifier-like potassium current could be explained by the activation of a novel muscarinic receptor subtype in which acetylcholine acts as the antagonist. Another possibility is that 4-AP activates IK(ACh) in a time- and voltage-dependent manner.

Differences in outward currents between neonatal and adult rabbit ventricular cells.

In adult rabbit ventricular preparations, action potential duration is significantly increased when stimulation frequency is increased from 0.1 to 1.0 Hz. In neonatal preparations, a similar change in stimulation frequency produced no significant increase in action potential duration. To identify the ionic basis for this difference, we studied different outward currents in single myocytes from papillary muscle and from epicardial tissue of adult and neonatal rabbits. The densities of the outward currents in neonatal cells were about one-half of the current density in adult cells. The density of the voltage-activated transient outward current (I(to1)) was smaller in cells from papillary muscle than in cells from epicardium in adult and newborn rabbits. We found major differences in the kinetic behavior of I(to1) between adult and neonatal cells: 1) the rate of apparent inactivation was faster in neonatal cells, and 2) the recovery from inactivation was significantly faster in neonatal cells, with a time constant of 113 vs. 1,356 ms. We propose that this marked difference in the recovery from inactivation of I(to1) is the basis for the difference in frequency dependence of action potential duration.