Biochemical and Biophysical Research Communications
Regular ArticleIP3-Activated Calcium-Permeable Channels in the Inside-Out Patches of Cultured Cerebellar Purkinje Cells
Abstract
Inositol-1,4,5,-trisphosphate (IP3)-activated calcium-permeable channels were recorded from inside-out patches of cultured cerebellar Purkinje cells. When 2-5 μM of IP3 was applied to the internal surface of the inside-out patches, inward Ba2+ currents were activated within 10 sec following the application in 11 out of 24 patches. In the presence of heparin (100 μg/ml), activation of Ba2+ currents by IP3 was inhibited. Unitary currents with different amplitudes and kinetics were observed; small and large unitary currents, and rapid fluctuations with various amplitudes. The small unitary currents (single channel conductance; 5.6 pS) were most frequent. Addition of inositol 1,3,4-trisphosphate (2-5 μM) slightly activated Ba2+ currents in 2 out of 10 patches, but the amount of the increment was much smaller than that produced by IP3. These results suggest a possibility that IP3 directly activates Ca2+-permeable channels in the plasma membrane of cerebellar Purkinje cells.
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Characterization of inositol-1,4,5-trisphosphate-gated channels in the plasma membrane of rat olfactory neurons
1999, Biophysical JournalIt is generally accepted that inositol-1,4,5-trisphosphate (InsP3) plays a role in olfactory transduction. However, the precise mode of action of InsP3 remains controversial. We have characterized the conductances activated by the addition of 10 μM InsP3 to excised patches of soma plasma membrane from rat olfactory neurons. InsP3 induced current fluctuations in 25 of 121 inside-out patches. These conductances could be classified into two groups according to the polarity of the current at a holding potential of +40 to +60 mV (with Ringer’s in the pipette and pseudointracellular solution in the bath). Conductances mediating outward currents could be further divided into large- (64 ± 4 pS, n = 4) and small- (16 ± 1.7 pS, n = 11) conductance channels. Both small- and large-conductance channels were nonspecific cation channels. The large-conductance channel displayed bursting behavior at +40 mV, with flickering increasing at negative holding potentials to the point where single-channel currents were no longer discernible. The small-conductance channel did not display flickering behavior. The conductance mediating inward currents at +40 to +60 mV reversed at +73 ± 4 mV (n = 4). The current traces displayed considerable fluctuations, and single-channel currents could not be discerned. The current fluctuations returned to baseline after removal of InsP3. The power density spectrum for the excess noise generated by InsP3 followed a 1/f dependence consistent with conductance fluctuations in the channel mediating this current, although other mechanisms are not excluded. These experiments demonstrate the presence of plasma membrane InsP3-gated channels of different ionic specificity in olfactory receptor cells.
Comparative effects of age and chronic low-level lead exposure on calcium mobilization from intracellular calcium stores in brain samples obtained from the neonatal and the adult rats
1997, Comparative Biochemistry and Physiology - C Pharmacology Toxicology and EndocrinologyThe effects of age and chronic low-level lead exposure were studied on (a) [3H]IP3 and [3H]Ry binding to their respective receptors in brain membranes and (b) Ca2+ release from internal Ca2+ stores in brain synaptosomes obtained from the neonatal and adult rats. [3H]IP3 and [3H]Ry binding sites in the control-adult membranes were greater than those in the control-neonatal membranes. [3H]IP3 bound to a single high-affinity site, IP3-R. Ca2+ decreased [3H]IP3 binding to its receptor. [3H]Ry bound to at least four subspecies of Ry-Rs. KCl and IP3 increased, but Ca2+ caused a biphasic affect on [3H]Ry binding in brain membranes. IP3 and caffeine both caused greater increase in [Ca2+]I in the adult synaptosomes than the neonatal synaptosomes. IP4 redistributed Ca2+ from the caffeine-sensitive pool to the IP3-sensitive pool. IP3 increased the caffeine-induced mobilization of Ca2+ in synaptosomes. Chronic low-level lead exposure decreased the binding of [3H]IP3 to its receptors in membranes, attenuated the IP3-induced Ca2+ mobilization in synaptosomes, abolished the IP4-induced redistribution of Ca2+ from Ry sensitive Ca2+ store to IP3-sensitive Ca2+ store, and attenuated the effects of IP3 on [Ca2+]I in caffeine stimulated synaptosomes. Lead exposure, however, did not affect [3H]Ry binding to Ry-R in membranes or the caffeine-induced increase in [Ca2+]I in synaptosomes. Chronic lead exposure protected IP3-R against Ca2+-induced inhibition in membranes. This protection was greater in the neonatal samples than the adult samples. This suggests that chronic low-level lead exposure down-regulated the IP3-induced Ca2+ mobilization in synaptosomes without effecting the caffeine-induced Ca2+ mobilization.
IP3-sensitive calcium channel
1997, Biomembranes: A Multi-Volume TreatiseIP3 is a second messenger that releases Ca2+ from the Ca2+ store site. cDNA cloning and sequencing of the IP3 receptor revealed the functional structure of the IP3R. IP3R has an IP3 binding domain of about 650 amino acids at the N-terminus. A regulatory (or coupling) domain lies at the middle part that links both the IP3-binding domain and the Ca2+ channel domain at the C-terminus. IP3-induced Ca2+ releasing activity is enhanced by ATP binding and by PKA phosphorylation. CaM kinase and PKC also phosphorylate IP3R. Ca2+ regulates the IP3-induced Ca2+ releasing activity in a bell-shape form. The channel domain has six membrane-spanning segments (M1–M6). A hydrophobic region between the M5 and M6 segments is considered to be a pore-forming region. Overall homology in the channel domains indicates that IP3R belongs to a family of voltage-sensitive and second messenger-gated channels. IP3R isoforms are produced from RNA splicing in the IP3-binding and regulatory domains. Each isoform is expressed in a region-specific and developmentally specific manner. Complementary DNA sequences of type 1, 2, and 3 IP3R are determined. These subtypes are expressed differentially: Type 1 IP3R is expressed at high level in the central nervous system, while type 2 and 3 expressions are observed in hematopoietic and lymphatic cell types.
Monoclonal antibody against IP3R blocked sperm-induced Ca2+ waves and oscillations in hamster egg. Antisense nucleotide injected in the Xenopus oocyte suppressed egg activation. These experiments show that IP3R is involved in Ca2+ waves and oscillations as well as egg activation.
Pharmacological modulators of the inositol 1,4,5-trisphosphate receptor
1995, NeuropharmacologyElevation of cytosolic calcium concentrations, induced by many neurotransmitters, plays a crucial role in neuronal function. Some neurotransmitters produce the second messenger InsP3 which activates an intracellular calcium channel (InsP3 receptor) usually located in the endoplasmic reticulum. This article undertakes a comprehensive survey of most pharmacological modulators of the InsP3 receptor so far reported. This review discusses in detail competitive antagonists, non-competitive antagonists and thiol reactive reagents, highlighting their modes of action and in some cases indicating drawbacks in their use.
Functional properties of intracellular calcium-release channels
1995, Current Opinion in NeurobiologyTwo major classes of intracellular calcium-release channels have been identified, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. These channels are the largest ion channels identified to date. Recent studies have established that approximately 90% of each of these proteins protrudes into the cytoplasm, presumably exposing many regulatory sites on the channel and allowing functional interactions with other cytoplasmic proteins. Current work is aimed at understanding the molecular mechanisms and cellular roles of these regulatory processes.
Calcium stores in neurons and glia
1994, Neuroscience