After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K+ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was −90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a “foot in the door” mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

Fenología de la gametogénesis, madurez de conceptáculos, fertilidad y embriogénesis en Durvillaea antarctica (Chamisso) Hariot (Phaeophyta, Durvillaeales)Phenology of gametogenesis, maturity of conceptacles, fertility and embryogenesis in Durvillaea antarctica (Chamisso) Hariot (Phaeophyta, Durvillaeales)

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca 2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.

The contribution of Ca 2+ entry through different voltage-activated Ca 2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K + depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-a (PKC-a), but not on Ca 2+ /calmodulin-dependent protein kinase nor cyclic AMPdependent protein kinase. Blockade of L-type Ca 2+ channels by 3 lM furnidipine, or blockade of N channels by 1 lM x-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 lM x-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca 2+ elevations elicited by 40 mM K + , showed that Ca 2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca 2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca 2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca 2+ in the cytosol rather than on activation of a given Ca 2+ channel subtype. The expression of L, N, P/Q, R and T subtypes of voltageactivated Ca 2+ channels (VACC) in neurons (Olivera et al. 1994;García et al. 2000) poses the interesting question of their specialization to control different cell functions. For instance, N and P/Q-type channels, which are predominantly found along the length of apical dendrites and in axon terminals that synapse on dendrites (Westenbroek et al. 1992), control the release of various neurotransmitters (Olivera et al. 1994;Wheeler et al. 1994;García et al. 2000). In contrast, L-type channels located on proximal dendrites and neuronal cell bodies Ahlijanian et al. 1990;Westenbroek et al. 1992;Waterman 1997;Westenbroek et al. 1998; Timmerman et al. 2002) have been associated with the regulation of gene expression and enzyme activity in cortical and hippocampal neurons (Murphy et al. 1991;Bading et al. 1993; Elliot et al. 1995;Westenbroek et al. 1995;Deisseroth et al. 1998).The particular segregation of Ca 2+ channel subtypes to dendrites, axon terminals, or somata facilitates their specialization to accomplish such specific functions in various neuronal cell types. However, specialization of the different