Rapid beta-adrenergic modulation of cardiac calcium channel currents by a fast G protein pathway

beta-Adrenergic agonists activate the G protein, Gs, which stimulates cardiac calcium currents by both cytoplasmic, indirect and membrane-delimited, direct pathways. To test whether beta-adrenergic agonists might use both pathways in the heart, isoproterenol was rapidly applied to cardiac myocytes, resulting in a biphasic increase in cardiac calcium channel currents that had time constants of 150 milliseconds and 36 seconds. beta-Adrenergic antagonists of a G protein inhibitor blocked both the fast and slow responses, whereas the adenylyl cyclase activator forskolin produced only the slow response. The presence of a fast pathway in the heart can explain what the slow pathway cannot account for: the ability of cardiac sympathetic nerves to change heart rate within a single beat.     

Development of two types of calcium channels in cultured mammalian hippocampal neurons

Calcium influx through voltage-gated membrane channels plays a crucial role in a variety of neuronal processes, including long-term potentiation and epileptogenesis in the mammalian cortex. Recent studies indicate that calcium channels in some cell types are heterogeneous. This heterogeneity has now been shown for calcium channels in mammalian cortical neurons. When dissociated embryonic hippocampal neurons from rat were grown in culture they first had only low voltage-activated, fully inactivating somatic calcium channels. These channels were metabolically stable and conducted calcium better than barium. Appearing later in conjunction with neurite outgrowth and eventually predominating in the dendrites, were high voltage-activated, slowly inactivating calcium channels. These were metabolically labile and more selective to barium than to calcium. Both types of calcium currents were reduced by classical calcium channel antagonists, but the low voltage-activated channels were more strongly blocked by the anticonvulsant drug phenytoin. These findings demonstrate the development and coexistence of two distinct types of calcium channels in mammalian cortical neurons.     

Splice variants of the alpha subunit of the G protein Gs activate both adenylyl cyclase and calcium channels

Signal transducing guanine nucleotide binding (G) proteins are heterotrimers with different alpha subunits that confer specificity for interactions with receptors and effectors. Eight to ten such G proteins couple a large number of receptors for hormones and neurotransmitters to at least eight different effectors. Although one G protein can interact with several receptors, a given G protein was thought to interact with but one effector. The recent finding that voltage-gated calcium channels are stimulated by purified Gs, which stimulates adenylyl cyclase, challenged this concept. However, purified Gs may have four distinct alpha-subunit polypeptides, produced by alternative splicing of messenger RNA. By using recombinant DNA techniques, three of the splice variants were synthesized in Escherichia coli and each variant was shown to stimulate both adenylyl cyclase and calcium channels. Thus, a single G protein alpha subunit may regulate more than one effector function.     

A G protein couples serotonin and GABAB receptors to the same channels in hippocampus

Both serotonin and the selective gamma-aminobutyric acidB (GABAB) agonist, baclofen, increase potassium (K+) conductance in hippocampal pyramidal cells. Although these agonists act on separate receptors, the potassium currents evoked by the agonists are not additive, indicating that the two receptors share the same potassium channels. Experiments with hydrolysis-resistant guanosine triphosphate (GTP) and guanosine diphosphate analogs and pertussis toxin indicate that the opening of the potassium channels by serotonin and GABAB receptors involves a pertussis toxin-sensitive GTP-binding (G) protein, which may directly couple the two receptors to the potassium channel.     

Modulation of calcium channels in cardiac and neuronal cells by an endogenous peptide

Calcium channels mediate the generation of action potentials, pacemaking, excitation-contraction coupling, and secretion and signal integration in muscle, secretory, and neuronal cells. The physiological regulation of the L-type calcium channel is thought to be mediated primarily by guanine nucleotide-binding proteins (G proteins). A low molecular weight endogenous peptide has been isolated and purified from rat brain. This peptide regulates up and down the cardiac and neuronal calcium channels, respectively. In cardiac myocytes, the peptide-induced enhancement of the L-type calcium current had a slow onset (half-time approximately 75 seconds), occurred via a G protein-independent mechanism, and could not be inhibited by alpha 1-adrenergic, beta-adrenergic, or angiotensin II blockers. In neuronal cells, on the other hand, the negative effect had a rapid onset (half-time less than 500 milliseconds) and was observed on both T-type and L-type calcium channels.     

Occult Drosophila calcium channels and twinning of calcium and voltage-activated potassium channels

In the membrane of the flight muscle cells of developing Drosophila a large calcium-sensitive potassium current, IKc, was found. It was present before the development of voltage-activated potassium channels and seems to be the first potassium current to develop in the membrane. Also present in these early cells were large numbers of occult (hidden) calcium channels, which remained inactive until the end of pupal development. These inactive calcium channels could be made to function by injecting adenosine triphosphate or ethyleneglycol tetraacetic acid into the early cells. IKc has kinetic properties resembling the later developing voltage-sensitive current IKv, and is distinct from the fast, transient calcium-dependent outward current IAc, which appears much later in development. IAc closely resembles the voltage-sensitive current IAv, also present in these cells. Thus, both of the voltage-sensitive potassium channel types, IAv and IKv, have similar calcium-sensitive counterparts, IAc and IKc, that are present in the same cells.     

Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory protein Gk

The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.     

Atrionatriuretic peptide transforms cardiac sodium channels into calcium-conducting channels

The atrionatriuretic peptide (ANP) is released from atrial cells in response to increased extracellular fluid volume and reduces sodium absorption by the kidney, thus reducing the blood volume. In this report, ANP suppressed the calcium and sodium currents in rat and guinea pig ventricular myocytes. The suppression of sodium current was caused by enhanced permeability of the sodium channel to calcium without significant changes in the kinetics or the tetrodotoxin sensitivity of the channel. Thus, ANP may regulate the sodium channel by altering its cationic selectivity site to calcium, thereby repressing the sodium current. The suppression of sodium and calcium channels and the resultant depressed excitability of the atrial cells may help to regulate ANP secretion.     

Voltage-dependent calcium channels in glial cells

The electrophysiological properties of glial cells were examined in primary culture in the presence of tetraethylammonium and Ba2+, a treatment that reduces K+ permeability of the membrane and enhances currents through voltage-dependent Ca2+ channels. Under these conditions, glial cells showed both spontaneous action potentials and action potentials evoked by the injections of current. These responses appear to represent entry of Ba2+ through Ca2+ channels because they were resistant to tetrodotoxin but were blocked by Mn2+ or Cd2+.     

Multiple calcium channels and neuronal function

Recent investigations have demonstrated that neurons have a number of different types of calcium channels, each with their own unique properties and pharmacology. These calcium channels may be important in the control of different aspects of nerve activity. Some of the possibilities can now be discussed.     

Voltage-dependent calcium channels in plant vacuoles

Free calcium (Ca(2+)) in the cytoplasm of plant cells is important for the regulation of many cellular processes and the transduction of stimuli. Control of cytoplasmic Ca(2+) involves the activity of pumps, carriers, and possibly ion channels. The patch-clamp technique was used to study Ca(2+) channels in the vacuole of sugar beet cells. Vacuolar currents showed inward rectification at negative potentials, with a single-channel conductance of 40 picosiemens and an open probability dependent on potential. Channels were inhibited by verapamil and lanthanum. These channels could participate in the regulation of cytoplasmic Ca(2+) by sequestering Ca(2+) inside the vacuole.     

A pertussis toxin-sensitive G protein in hippocampal long-term potentiation

High-frequency (tetanic) stimulation of presynaptic nerve tracts in the hippocampal region of the brain can lead to long-term synaptic potentiation (LTP). Pertussis toxin prevented the development of tetanus-induced LTP in the stratum radiatum-CA1 synaptic system of rat hippocampal slices, indicating that a guanosine triphosphate-binding protein (G protein) may be required for the initiation of LTP. This G protein may be located at a site distinct from the postsynaptic neuron (that is, in presynaptic terminals or glial cells) since maximal activation of CA1 neuronal G proteins by intracellular injection of guanosine-5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of guanosine 5'-triphosphate, did not occlude LTP.     

TRPM4 regulates calcium oscillations after T cell activation

TRPM4 has recently been described as a calcium-activated nonselective (CAN) cation channel that mediates membrane depolarization. However, the functional importance of TRPM4 in the context of calcium (Ca2+) signaling and its effect on cellular responses are not known. Here, the molecular inhibition of endogenous TRPM4 in T cells was shown to suppress TRPM4 currents, with a profound influence on receptor-mediated Ca2+ mobilization. Agonist-mediated oscillations in intracellular Ca2+ concentration ([Ca2+]i), which are driven by store-operated Ca2+ influx, were transformed into a sustained elevation in [Ca2+]i. This increase in Ca2+ influx enhanced interleukin-2 production. Thus, TRPM4-mediated depolarization modulates Ca2+ oscillations, with downstream effects on cytokine production in T lymphocytes.     

Actomyosin-like protein isolated from mammalian brain

A protein with characteristics similar to actomyosin has been isolated from whole brain of rat and cat. It is soluble in 0.6 molar potassium chloride and insoluble in 0.1 molar potassium chloride. It superprecipitates with magnesium ions and adenosine triphosphate. It has adenosine triphosphatase activity stimulated by either magnesium or calcium ions. Both superprecipitation and adenosine triphosphatase activity are inhibited by p-chloromercuribenzoate and Mersalyl but not by ouabain.     

Calcium-sensitive inactivation in the gating of single calcium channels

Voltage-activated calcium channels open and close, or gate, according to molecular transition rates that are regulated by transmembrane voltage and neurotransmitters. Here evidence for the control of gating by calcium was found in electrophysiological records of single, L-type calcium channels in heart cells. Conditional open probability analysis revealed that calcium entry during the opening of a single channel produces alterations in gating transition rates that evolve over the course of hundreds of milliseconds. Such alteration of calcium-channel gating by entry of a favored permeant ion provides a mechanism for the short-term modulation of single-ion channels.     

Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells

Arachidonic acid, as well as fatty acids that are not substrates for cyclooxygenase and lipoxygenase enzymes, activated a specific type of potassium channel in freshly dissociated smooth muscle cells. Activation occurred in excised membrane patches in the absence of calcium and all nucleotides. Therefore signal transduction pathways that require such soluble factors, including the NADPH-dependent cytochrome P450 pathway, do not mediate the response. Thus, fatty acids directly activate potassium channels and so may constitute a class of signal molecules that regulate ion channels.