ABSTRACT
Exocytosis proceeds by either full fusion or 'kiss-and-run' between vesicle and plasma membrane. Switching between these two modes permits the cell to regulate the kinetics and amount of secretion. Here we show that ATP receptor activation reduces secretion downstream from cytosolic Ca2+ elevation in rat adrenal chromaffin cells. This reduction is mediated by activation of a pertussis toxin-sensitive G(i/o) protein, leading to activation of G(betagamma) subunits, which promote the 'kiss-and-run' mode by reducing the total open time of the fusion pore during a vesicle fusion event. Furthermore, parallel activation of the muscarinic acetylcholine receptor removes the inhibitory effects of ATP on secretion. This is mediated by a G(q) pathway through protein kinase C activation. The inhibitory effects of ATP and its reversal by protein kinase C activation are also shared by opioids and somatostatin. Thus, a variety of G protein pathways exist to modulate Ca2+-evoked secretion at specific steps in fusion pore formation.
Subject(s)
Chromaffin Cells/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Protein Kinase C/metabolism , Receptors, G-Protein-Coupled/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Action Potentials/radiation effects , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Adrenal Medulla/cytology , Analgesics, Opioid/pharmacology , Animals , Calcium/metabolism , Cells, Cultured , Chromaffin Cells/drug effects , Dose-Response Relationship, Radiation , Drug Interactions , Dynamins/pharmacology , Electric Stimulation/methods , Electrochemistry/methods , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , GTP-Binding Protein beta Subunits/pharmacology , Ionomycin/pharmacology , Ionophores/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/radiation effects , Muscarine/pharmacology , Neural Inhibition/drug effects , Neural Inhibition/physiology , Patch-Clamp Techniques/methods , Pertussis Toxin/pharmacology , Potassium Chloride/pharmacology , Protein Kinases/pharmacology , Rats , Recombinant Fusion Proteins/pharmacology , Somatostatin/pharmacology , Thionucleotides/pharmacologyABSTRACT
The hyperpolarization-activated cation channels (I(h)) play a distinct role in rhythmic activities in a variety of tissues, including neurons and cardiac cells. In the present study, we investigated whether Ca(2+) can permeate through the hyperpolarization-activated pacemaker channels (HCN) expressed in HEK293 cells and I(h) channels in dorsal root ganglion (DRG) neurons. Using combined measurements of whole-cell currents and fura-2 Ca(2+) imaging, we found that there is a Ca(2+) influx in proportion to I(h) induced by hyperpolarization in HEK293 cells. The I(h) channel blockers Cs(+) and ZD7288 inhibit both HCN current and Ca(2+) influx. Measurements of the fractional Ca(2+) current showed that it constitutes 0.60 +/- 0.02% of the net inward current through HCN4 at -120 mV. This fractional current is similar to that of the low Ca(2+)-permeable AMPA-R (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) channels in Purkinje neurons. In DRG neurons, activation of I(h) for 30 s also resulted in a Ca(2+) influx and an elevated action potential-induced secretion, as assayed by the increase in membrane capacitance. These results suggest a functional significance for I(h) channels in modulating neuronal secretion by permitting Ca(2+) influx at negative membrane potentials.
Subject(s)
Calcium/metabolism , Ion Channels/metabolism , Neurons/metabolism , Animals , Cell Line , Humans , Long-Term Potentiation , Neurons/physiology , Patch-Clamp Techniques , Rats , Rats, Wistar , Spectrometry, FluorescenceABSTRACT
A comparative study was carried out on the inactivation of Na+ channels in two types of endocrine cells in rats, beta-cells and adrenal chromaffin cells (ACCs), using patch-clamp techniques. The beta-cells were very sensitive to hyperpolarization; the Na+ currents increased ninefold when the holding potential was shifted from -70 mV to -120 mV. ACCs were not sensitive to hyperpolarization. The half-inactivation voltages were -90 mV (rat beta-cells) and -62 mV (ACCs). The time constant for recovery from inactivation at -70 mV was 10.5 times slower in beta-cells (60 ms) than in ACCs (5.7 ms). The rate of Na+-channel inactivation at physiological resting potential was more than three times slower in beta-cells than in ACCs. Na+ influx through Na+ channels had no effect on the secretory machinery in rat beta-cells. However, these 'silent Na+ channels' could contribute to the generation of action potentials in some conditions, such as when the cell is hyperpolarized. It is concluded that the fractional availability of Na+ channels in beta-cells at a holding potential of -70 mV is about 15 % of that in ACCs. This value in rat beta-cells is larger than that observed in mouse (0 %), but is smaller than those observed in human or dog (90 %).
Subject(s)
Adrenal Glands/metabolism , Chromaffin Cells/metabolism , Islets of Langerhans/drug effects , Sodium Channel Blockers/pharmacology , Adrenal Glands/cytology , Adrenal Glands/drug effects , Animals , Cells, Cultured , Chromaffin Cells/drug effects , Electrophysiology , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice , Patch-Clamp Techniques , Rats , Rats, Wistar , Tolbutamide/pharmacologyABSTRACT
A novel conotoxin, kappa-conotoxin (kappa-BtX), has been purified and characterized from the venom of a worm-hunting cone snail, Conus betulinus. The toxin, with four disulfide bonds, shares no sequence homology with any other conotoxins. Based on a partial amino acid sequence, its cDNA was cloned and sequenced. The deduced sequence consists of a 26-residue putative signal peptide, a 31-residue mature toxin, and a 13-residue extra peptide at the C terminus. The extra peptide is cleaved off by proteinase post-processing. All three Glu residues are gamma-carboxylated, one of the two Pro residues is hydroxylated at position 27, and its C-terminal residue is Pro-amidated. The monoisotopic mass of the toxin is 3569.0 Da. Electrophysiological experiments show that: 1) among voltage-gated channels, kappa-BtX is a specific modulator of K(+) channels; 2) among the K channels, kappa-BtX specifically up-modulates the Ca(2+)- and voltage-sensitive BK channels (252 +/- 47%); 3) its EC(50) is 0.7 nm with a single binding site (Hill = 0.88); 4) the time constant of wash-out is 8.3 s; and 5) kappa-BtX has no effect on single channel conductance, but increases the open probability of BK channels. It is concluded that kappa-BtX is a novel specific biotoxin against BK channels.
