Time Course of the Increase in the Myocardial Slow Inward Current after a Photochemically Generated Concentration Jump of Intracellular cAMP

Voltage-clamped atrial trabeculae from bullfrog hearts were exposed to membrane-permeant photolyzable o-nitrobenzyl esters of cAMP and cGMP. UV flashes produced intracellular concentration jumps of cAMP or cGMP. With the cAMP derivative, flashes resulted in an increased slow inward current (Isi), producing a broadened action potential. The Isi reached a maximum 10-30 sec after the flash and decreased over the next 60-300 sec. The first increases were observable within 150 msec; this value is an upper limit imposed by the instrumentation. Responses to flashes lasted longer at higher drug concentrations and in the presence of the phosphodiesterase inhibitor papaverine; effects of flashes developed and decreased faster at higher temperature. Although the amplitude of the Isi was increased, its waveform and voltage sensitivity were not affected. Intracellular concentration jumps of cAMP failed to affect the muscarinic K+ conductance. There were no observable effects of cGMP concentration jumps. The data confirm (i) that cAMP regulates the Isi and (ii) that the 5- to 10-sec delay between application of ß-agonists and the onset of positive inotropic effects, observed in previous studies, has been correctly ascribed to events prior to the interaction between cAMP and protein kinase

A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart

These experiments employ the photoisomerizable compound, 3,3'-bis- [alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans- Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy

Ca channels induced in Xenopus oocytes by rat brain mRNA

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2–5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. I_(Ba) in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 ± 4 nA, n = 21). In contrast, injected oocytes showed a peak I_(Ba) of 339 ± 42 nA (n = 33). The threshold for activation of I_(Ba) was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (τ = 650 ± 50 msec at +20 mV). The maintained current was 67 ± 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward I_(Ba) could be observed in BaCl₂ solutions at potentials positive to E_(Cl) and also in Na-free salines, indicating that neither Cl⁻ nor Na⁺ was carrying the inward current. Although I_(Ba) displayed voltage- independent blockade by Cd (50% inhibition at 6 µM), the peptide Ca channel antagonist, ω-CgTX (1 µM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 µM, or when cells were held at -40 mV) or agonist Bay K-8644. However, I_(Ba) was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT₂). In contrast, use of forskolin to activate protein kinase A did not alter I_(Ba). However, experiments in the presence of Cd revealed that forskolin decreased I_K. Ca channels produced by rat brain mRNA were thus in contrast to the nifedipine-sensitive, Bay K-8644- and forskolin-enhanced Ca channels observed after injection of rat heart mRNA (Dascal et al., 1986)

Ca channels induced in Xenopus oocytes by rat brain mRNA

RNA was isolated from brains of 16-d-old rats and poly(A) samples were injected into stage V and VI oocytes. After allowing 2–5 d for expression, most oocytes were exposed to medium in which the K had been replaced by Cs for 24 hr prior to recording. Ba currents were usually measured in Cl-free Ba-methanesulfonate saline. I_(Ba) in noninjected oocytes was often undetectable, but ranged up to 50 nA (22 ± 4 nA, n = 21). In contrast, injected oocytes showed a peak I_(Ba) of 339 ± 42 nA (n = 33). The threshold for activation of I_(Ba) was -40 mV, with peak currents at +10 to +20 mV. After a peak, currents decayed to a nearly steady level along a single-exponential time course (τ = 650 ± 50 msec at +20 mV). The maintained current was 67 ± 6% (n = 9) of the early peak amplitude. A prepulse duration of 5 sec was needed to examine the inactivation of barium currents in injected oocytes. The inward I_(Ba) could be observed in BaCl₂ solutions at potentials positive to E_(Cl) and also in Na-free salines, indicating that neither Cl⁻ nor Na⁺ was carrying the inward current. Although I_(Ba) displayed voltage- independent blockade by Cd (50% inhibition at 6 µM), the peptide Ca channel antagonist, ω-CgTX (1 µM), and the organic Ca channel-blocking agents (verapamil, compound W-7, and nifedipine) were uniformly ineffective. No effects were observed with the dihydropyridine antagonist nifedipine (even at 10 µM, or when cells were held at -40 mV) or agonist Bay K-8644. However, I_(Ba) was enhanced via activation of protein kinase C with 4-beta-phorbol dibutyrate (PBT₂). In contrast, use of forskolin to activate protein kinase A did not alter I_(Ba). However, experiments in the presence of Cd revealed that forskolin decreased I_K. Ca channels produced by rat brain mRNA were thus in contrast to the nifedipine-sensitive, Bay K-8644- and forskolin-enhanced Ca channels observed after injection of rat heart mRNA (Dascal et al., 1986)

Aldosterone increases T-type calcium channel expression and in vitro beating frequency in neonatal rat cardiomyocytes

Objective: Although aldosterone has been implicated in the pathogenesis of cardiac hypertrophy and heart failure, its cellular mechanism of action on cardiomyocyte function is not yet completely elucidated. This study was designed to investigate the effect of aldosterone on calcium channel expression and cardiomyocyte contraction frequency. Methods: Cultured neonatal rat ventricular cardiomyocytes were stimulated in vitro with 1 μmol/L aldosterone for 24 h. Calcium currents were then measured with the patch clamp technique, while calcium channel expression was assessed by real-time RT-PCR. Results: In the present study, we show that aldosterone increases Ca2+ currents by inducing channel expression. Indeed, aldosterone led to a substantial increase of L- and T-type Ca2+ current amplitudes, and we found a concomitant 55% increase of the mRNA coding for α1C and β2 subunits of cardiac L channels. Although T-type currents were relatively small under control conditions, they increased 4-fold and T channel α1H isoform expression rose in the same proportion after aldosterone treatment. Because T channels have been implicated in the modulation of membrane electrical activity, we investigated whether aldosterone affects the beating frequency of isolated cardiomyocytes. In fact, aldosterone dose-dependently increased the spontaneous beating frequency more than 4-fold. This effect of aldosterone was prevented by actinomycin D and spironolactone and reduced by RU486, suggesting a mixed mineralocorticoid/glucocorticoid receptor-dependent transcriptional mechanism. Moreover, inhibition of T currents with Ni2+ or mibefradil significantly reduced beating frequency towards control values, while conditions affecting L-type currents completely blocked contractions. Conclusion: Aldosterone modulates the expression of cardiac voltage-operated Ca2+ channels and accelerates beating in cultured neonatal rat ventricular myocytes. This chronotropic action of aldosterone appears to be linked to increased T channel activity and could contribute to the deleterious effect of an excess of this steroid in vivo on cardiac functio

vitro analog of operant conditioning in Aplysia. II. Modifications of the functional dynamics of an identified neuron contribute to motor pattern selection

Previously, an analog of operant conditioning was developed using the buccal ganglia of Aplysia, the probabilistic occurrences of a specific motor pattern (i.e., pattern I), a contingent reinforcement (i.e., stimulation of the esophageal nerve), and monotonic stimulation of a peripheral nerve (i.e., n.2,3). This analog expressed a key feature of operant conditioning (i.e., selective enhancement of the probability of occurrence of a designated motor pattern by contingent reinforcement). In addition, the training induced changes in the dynamical properties of neuron B51, an element of the buccal central pattern generator. To gain insights into the neuronal mechanisms that mediate features of operant conditioning, the present study identified a neuronal element that was critically involved in the selective enhancement of pattern I. We found that bursting activity in cell B51 contributed significantly to the expression of pattern I and that changes in the dynamical properties of this cell were associated with the selective enhancement of pattern I. These changes could be induced by an explicit association of reinforcement with random depolarization of B51. No stimulation of n.2,3 was required. These results indicate that the selection of a designated motor pattern by contingent reinforcement and the underlying neuronal plasticity resulted from the association of reinforcement with a component of central neuronal activity that contributes to a specific motor pattern. The sensory stimulus that allows for occurrences of different motor acts may not be critical for induction of plasticity that mediates the selection of a motor output by contingent reinforcement in operant conditioning

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel α-subunit expressed in mammalian cells

The rat brain IIA Na⁺ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na⁺ channel α-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (~10 channels/ µm² in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC₃H1 cells and failed to express in Ltk⁻ cells. However, voltage-dependent Drosophila Shaker H4 K⁺ channels and Escherichia coli β-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na⁺ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na⁺ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na⁺ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell- specific posttranslational modifications. IIA channels were blocked by ~90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX- resistant Na⁺ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence

The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic β-cell line

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic β-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I–II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels