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

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

New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations

The cyclic nucleotides cyclic AMP and cyclic GMP are important intracellular messengers mediating the responses to neurotransmitters and neurohormones and regulating cellular function over a wide range of time scales. Despite the widespread acceptance of this second messenger mechanism in many systems, much remains unknown about their mechanism of action, except that such events are associated with increases or decreases in intracellular cyclic nucleotides. Quantitative descriptions of cyclic nucleotide-dependent processes are hampered by the absence of a means by which intracellular cyclic nucleotide concentrations can be accurately controlled. We have now designed, synthesized and characterized new, substituted photolabile cyclic nucleotide analogues, the 4,5-dimethoxy-2-nitrobenzyl esters of cyclic AMP and cyclic GMP (Fig. 1), which are physiologically inert before irradiation and which liberate free cyclic AMP or cyclic GMP on absorption of a photon. The thermal properties and photolysis rates and efficiencies of light-induced release of cyclic nucleotides from these analogues are more favourable than for the simple o-nitrobenzyl derivatives used previously. These molecules should permit intracellular ‘concentration jumps’ of cyclic AMP or cyclic GMP to be produced in cells under physiological investigation with spatial and temporal resolution unmatched by conventional techniques

New photoactivatable cyclic nucleotides produce intracellular jumps in cyclic AMP and cyclic GMP concentrations

The cyclic nucleotides cyclic AMP and cyclic GMP are important intracellular messengers mediating the responses to neurotransmitters and neurohormones and regulating cellular function over a wide range of time scales. Despite the widespread acceptance of this second messenger mechanism in many systems, much remains unknown about their mechanism of action, except that such events are associated with increases or decreases in intracellular cyclic nucleotides. Quantitative descriptions of cyclic nucleotide-dependent processes are hampered by the absence of a means by which intracellular cyclic nucleotide concentrations can be accurately controlled. We have now designed, synthesized and characterized new, substituted photolabile cyclic nucleotide analogues, the 4,5-dimethoxy-2-nitrobenzyl esters of cyclic AMP and cyclic GMP (Fig. 1), which are physiologically inert before irradiation and which liberate free cyclic AMP or cyclic GMP on absorption of a photon. The thermal properties and photolysis rates and efficiencies of light-induced release of cyclic nucleotides from these analogues are more favourable than for the simple o-nitrobenzyl derivatives used previously. These molecules should permit intracellular ‘concentration jumps’ of cyclic AMP or cyclic GMP to be produced in cells under physiological investigation with spatial and temporal resolution unmatched by conventional techniques

Photochemically produced intracellular concentration jumps of cAMP mimic the effects of catecholamines on excitation-contraction coupling in frog atrial fibers

Previously, we reported that concentration jumps of cAMP produced by light flashes in the presence of a photosensitive analogue of cAMP increase the amplitude of the slow inward current (I_(si)) in isolated bullfrog atrial trabeculae (Nargeot et al. 1983). Here, using newly designed photolabile cyclic nucleotides (Nerbonne et al. 1984a), we have examined the effects of intracellular concentration jumps of cAMP and cGMP on excitation-contraction coupling in frog heart. Concentration jumps of cAMP increase the amplitude and the duration of action potentials, increase I_(si) and twitch tension. Following single flashes, maximum responses are observed in 10–30 s and recovery times are 30–120 s. The time courses of the cAMP-induced increases in I_(si) and phasic tension amplitudes are parallel, implying a direct correlation between Ca²⁺ influx through the slow channels and the development of phasic tension. Although the amplitudes are increased severalfold, cAMP jumps do not measurably alter the kinetics or voltage dependences of the current or tension. cAMP concentration jumps increase the delayed K⁺ current (I_K) and decrease tonic tension; relaxation of contraction is not, however, influenced by cAMP jumps. Concentration jumps of cGMP, on the other hand, have no measurable effects on the action potential, I_(si), I_K or tension in this preparation

Photochemically produced intracellular concentration jumps of cAMP mimic the effects of catecholamines on excitation-contraction coupling in frog atrial fibers

Previously, we reported that concentration jumps of cAMP produced by light flashes in the presence of a photosensitive analogue of cAMP increase the amplitude of the slow inward current (I_(si)) in isolated bullfrog atrial trabeculae (Nargeot et al. 1983). Here, using newly designed photolabile cyclic nucleotides (Nerbonne et al. 1984a), we have examined the effects of intracellular concentration jumps of cAMP and cGMP on excitation-contraction coupling in frog heart. Concentration jumps of cAMP increase the amplitude and the duration of action potentials, increase I_(si) and twitch tension. Following single flashes, maximum responses are observed in 10–30 s and recovery times are 30–120 s. The time courses of the cAMP-induced increases in I_(si) and phasic tension amplitudes are parallel, implying a direct correlation between Ca²⁺ influx through the slow channels and the development of phasic tension. Although the amplitudes are increased severalfold, cAMP jumps do not measurably alter the kinetics or voltage dependences of the current or tension. cAMP concentration jumps increase the delayed K⁺ current (I_K) and decrease tonic tension; relaxation of contraction is not, however, influenced by cAMP jumps. Concentration jumps of cGMP, on the other hand, have no measurable effects on the action potential, I_(si), I_K or tension in this preparation