L-type calcium channels mediate transmitter release in isolated, wide-field retinal amacrine cells

Transmitter release in neurons is triggered by intracellular Ca2+ increase via the opening of voltage-gated Ca2+ channels. Here we investigated the voltage-gated Ca2+ channels in wide-field amacrine cells (WFACs) isolated from the white-bass retina that are functionally coupled to transmitter release. We monitored transmitter release through the measurement of the membrane capacitance (Cm). We found that 500-ms long depolarizations of WFACs from −70 mV to 0 mV elicited about a 6% transient increase in the Cm or membrane surface area. This Cm jump could be eliminated either by intracellular perfusion with 10 mM BAPTA or by extracellular application of 4 mM cobalt. WFACs possess N-type and L-type voltage-gated Ca2+ channels. Depolarization-evoked Cm increases were unaffected by the specific N-type channel blocker ω-conotoxin GVIA, but they were markedly reduced by the L-type blocker diltiazem, suggesting a role for the L-type channel in synaptic transmission. Further supporting this notion, in WFACs the synaptic protein syntaxin always colocalized with the pore-forming subunit of the retinal specific L-type channels (CaV1.4 or α1F), but never with that of the N-type channels (CaV2.2 or α1B).

ATP-induced rise in apamin-sensitive Ca(2+)-dependent K+ conductance in adult rat hepatocytes

Exogenous ATP-induced transient outward currents (IATP) were investigated in isolated adult rat hepatocytes using conventional whole cell patch and nystatin perforated patch recording modes. The IATP increased in a sigmoidal fashion with an increase in ATP concentration, where the half-maximal concentration was 1.4 microM. The order of current potency was 2-methylthio-ATP > or = UTP = ATP > > alpha, beta-methylene-ATP. IATP was depressed in a concentration-dependent manner by suramin and apamin. IATP reversed its direction at the K+ equilibrium potential. IATP occurred easily in hepatocytes obtained from female rats weighing > 250 g. Removal of extracellular Ca2+ had no effect on the peak amplitude of IATP, but thapsigargin abolished it. Intracellular perfusion with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, heparin, guanosine 5'-O-(3-thiotriphosphate), or neomycin also abolished IATP. Pretreatment with pertussis toxin or calmodulin antagonists had no effect on IATP. It was concluded that ATP binding to both P2Y and P2U purinoceptors coupled to G protein may raise apaminsensitive Ca(2+)-dependent K+ conductance via a phospholipase C-inositol trisphosphate-Ca2+ signaling pathway.

G alpha o1 decapeptide modulates the hippocampal 5-HT1A potassium current

1. The serotonin1A (5-HT1A) receptor is coupled to an inwardly rectifying potassium current (IKir) via a G protein. The identity of the G-protein subtype was investigated with 2 10-amino acid peptides derived from the carboxyl (C) terminus of the alpha-subunits of the Go1 and Gi2 proteins (G alpha o1 and G alpha i2). The synthetic decapeptides were applied by intracellular perfusion during whole cell recording from dentate granule cells in the hippocampal slice preparation. 2. Bath application of 5-HT produced an IKir, which was blocked by the selective 5-HT1A receptor antagonist, pindobind5-HT1A. The G alpha o1 peptide inhibited the 5-HT1A IKir by 60 +/- 7% (mean +/- SE; t = 30 min), whereas the G alpha i2 peptide had no effect. The G alpha o1 peptide produced a slowly developing outward current that was not observed in the absence of peptide or in the presence of the G alpha i2 peptide. 3. The results indicate that G alpha o1 and not G alpha i2 modulates the 5-HT1A IKir in hippocampal granule cells. They also suggest that G alpha o1 occludes the 5-HT1A response by direct activation of the IKir. The intracellular perfusion of synthetic G alpha peptides provides a new approach to identify the G-protein subtype(s) in a receptor-mediated electrophysiological response.

Suppression of a Ca2+ current by NMDA and intracellular Ca2+ in acutely isolated hippocampal neurons

1. Ca2+ current was examined in acutely isolated hippocampal cells with the use of whole cell voltage-clamp recording and under continuous intracellular perfusion. A persistent Ca2+ current was activated by depolarization to -10 mV from a holding potential of -50 mV. 2. The persistent Ca2+ current was suppressed upon a wash out of the intracellular Mg(2+)-ATP. Adenosine 3',5'-cyclic monophosphate (cAMP) introduced intracellularly potentiated the Ca2+ current, and kinase A inhibitor blocked the current. 3. Reversible suppression of the persistent Ca2+ current was also observed by elevating intracellular Ca2+. This Ca(2+)-dependent suppression was retarded by the addition of a phosphatase inhibitor, okadaic acid, to the intracellular solution. 4. N-methyl-D-aspartate (NMDA) elicited inward current (NMDA response) in the isolated cells. The persistent Ca2+ current was transiently suppressed after the NMDA response. Suppression of the Ca2+ current by NMDA was reduced when intracellular Ca2+ buffering capacity was increased by increasing the concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) from a concentration of 1-10 mM. 5. Substitution of ATP in the intracellular solution with ATP-gamma-S or the addition of okadaic acid to the intracellular solution reduced the suppressive effect of NMDA on the Ca2+ current. 6. The results suggest that the persistent Ca2+ current in the hippocampal cells is maintained by a kinase A-mediated phosphorylation. Increases in the intracellular Ca2+ concentration suppressed the Ca2+ current via a mechanism involving a phosphatase. Ca2+ entry through the NMDA receptor channel suppressed the Ca2+ channel by activating the phosphatase.