Search

Lateral diffusion on the neuronal plasma membrane of the AMPA-type glutamate receptor (AMPAR) serves an important role in synaptic plasticity. We investigated the role of the secreted glycoprotein Noelin1 (Olfactomedin-1 or Pancortin) in AMPAR lateral mobility and its dependence on the extracellular matrix (ECM). We found that Noelin1 interacts with the AMPAR with high affinity, however, without affecting rise- and decay time and desensitization properties. Noelin1 co-localizes with synaptic and extra-synaptic AMPARs and is expressed at synapses in an activity-dependent manner. Single-particle tracking shows that Noelin1 reduces lateral mobility of both synaptic and extra-synaptic GluA1-containing receptors and affects short-term plasticity. While the ECM does not constrain the synaptic pool of AMPARs and acts only extrasynaptically, Noelin1 contributes to synaptic potentiation by limiting AMPAR mobility at synaptic sites. This is the first evidence for the role of a secreted AMPAR-interacting protein on mobility of GluA1-containing receptors and synaptic plasticity.

Dopamine is a known inhibitor of pituitary melanotropic cells. It reduces Ca2+influx by hyperpolarizing the cell membrane and by modulating high- and low-voltage-activated (HVA and LVA) Ca2+channels. As a result, dopamine reduces the hormonal output of the cell. However, it is unknown how dopamine affects each of the four different HVA Ca2+channel types individually. Moreover, it is unknown whether dopamine interacts with exocytosis independent of Ca2+channels. Here we show that dopamine differentially modulates the HVA Ca2+channels and that it affects the stimulus-secretion coupling through a direct effect on the exocytotic machinery. Sustained L- and P-type Ba2+currents are reduced in amplitude and inactivating N- and Q-type currents acquire different activation and inactivation kinetics in the presence of dopamine. The Q-type current shows slow activation, which is a hallmark for direct G-protein modulation. We used membrane capacitance measurements to monitor exocytosis. Surprisingly, we find that the amount of exocytosis per step depolarization is not diminished by dopamine despite the reduction in Ca2+current. To test whether dopamine affects the release machinery downstream of Ca2+entry, we stimulated exocytosis by dialyzing cells with buffered high-Ca2+solutions. Dopamine increased the amount and the rate of exocytosis. In the first 90 s, the rate of secretion was increased two- to threefold, but it was normalized again at 180 s, suggesting that predominantly vesicles that fuse early in the exocytotic phase are modulated by dopamine. Thus while Ca2+channels are inhibited by dopamine, the exocytotic machinery downstream of Ca2+influx is sensitized. As a result, release is more effectively stimulated by Ca2+influx during dopamine inhibition.

The vasopressin/oxytocin-related neuropeptide Lys-conopressin activates two pacemaker currents in central neurons of the mollusk Lymnaea stagnalis. A high-voltage–activated current (I-HVA) is activated at potentials greater than −40 mV and resembles pacemaker currents found in many molluscan neurons. A low-voltage–activated current (I-LVA) activates throughout the range of −90 to 0 mV. Based on sequence homologies,Lymnaeaconopressin receptors are thought to couple to Q-type G proteins and protein kinase C (PKC). Alternatively, agonist-induced pacemaker currents in molluscan neurons have traditionally been attributed to cAMP-dependent protein kinase (PKA) activation. Accordingly, this study aimed at resolving possible involvement of cAMP/PKA and diacylglycerol/PKC in the conopressin response. Injection of cAMP into anterior lobe neurons induced a slow inward current with a voltage dependence resembling that ofILVA(and notIHVA). However, lack of effect of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and the absence of cross-desensitization between cAMP and conopressin suggest that neither current is dependent on intracellular cAMP. The PKC-activating phorbol ester 12-O-tetradecanoylphorbol 13-acetate (but not inactive phorbol 12-myristate 13-acetate) mimicked activation ofIHVA, but notILVA, and occluded subsequent responses to conopressin. Activation ofIHVAwas blocked by general protein kinase inhibitors and the PKC-inhibitor GF-109203X. Modulation of the calcium buffering capacity of the pipette medium did not affect the conopressin response, suggesting that calcium dynamics are not of major importance. We conclude that conopressin activates the ion channels carrying ILVAandIHVAthrough different second-messenger cascades and that PKC-dependent phosphorylation underlies activation of IHVA.

The light green cells (LGCs) are a group of identified central neurons in the pond snail, Lymnaea stagnalis, that produce a number of insulin-related peptides. Freshly dissociated LGCs are activated by physiological concentrations of extracellular glucose. The response to glucose consists of a slow depolarization, which, at concentrations of 1 mM or more, rapidly induces regular spiking activity. The response persists during prolonged application of glucose but is completely reversed upon washing. The threshold concentration is 0.2 mM; the maximal effect occurs at 5 mM. In LGCs in central nervous system preparations kept in organ culture for 16–24 h, glucose causes a similar depolarization, which may lead to spiking activity. In freshly isolated preparations, which have very inexcitable LGCs, no direct response to glucose was seen. The response is specific to the LGCs: no other central neurons in Lymnaea showed consistent responses. The glucose response is evoked by d-glucose and the non-metabolized analogue 2-deoxy-d-glucose, but not by related hexoses, including l-glucose, nor pentoses or disaccharides. The response is not affected by interfering with the glucose metabolism, nor is the response mimicked by the metabolite d-glyceraldehyde or by injection of glucose. This suggests that glucose metabolites are not involved in the response. The glucose response depends on the presence of extracellular Na+ and is blocked by phlorizin, which specifically inhibits Na+-coupled glucose transport. This suggests that the response is due to activation of an electrogenic Na+-coupled glucose transporter.

We describe a new peptide conotoxin affecting sodium current inactivation, that competes on binding with δ-conotoxin TxVIA (δTxVIA). The amino acid sequence of the new toxin, designated conotoxin NgVIA (NgVIA), is SKCFSOGTFCGIKOGLCCSVRCFSLFCISFE (where O is trans-4-hydroxyproline). The primary structure of NgVIA has an identical cysteine framework and similar hydrophobicity as δTxVIA but differs in its net charge. NgVIA competes with δTxVIA on binding to rat brain synaptosomes and molluscan central nervous system and strongly inhibits sodium current inactivation in snail neurons, as does δTxVIA. In contrast to δTxVIA, NgVIA is a potent paralytic toxin in vertebrate systems, its binding appears to be voltage-dependent, and it synergically increases veratridine-induced sodium influx to rat brain synaptosomes. δTxVIA acts as a partial antagonist to NgVIA in rat brain in vivo. NgVIA appears to act via a receptor site distinct from that of δTxVIA but similar to that of Conus striatustoxin. This new toxin provides a lead for structure-function relationship studies in the δ-conotoxins and will enable analysis of the functional significance of this complex of receptor sites in gating mechanisms of sodium channels.

Dopamine hyperpolarizes growth hormone-producing cells (GHC) in the CNS of Lymanea stagnalis. This effect of dopamine was mimicked by the D-2 receptor agonist LY 141865 and antagonized by the D-2 receptor antagonists (−) sulpiride and YM 09151-2. SKF 38393, a selective D-1 receptor aagonist, increased the excitability of the GHC. This effect was mimicked by intracellular injection of cyclic AMP andantonized by the D-1 receptor antagonist SCH23390. Dopamine (in the presence of (−)-sulpride) also increased the excitability of the GHC. It is concluded that both a D-1 and a D-2 receptor regulate the electrical activity of the GHC in the CNS of Lymnaea stagnalis.