Biomedical subjects

Eduardo Rojas

Publications and source records attributed to Eduardo Rojas.

5 recordsLinked to original sources

PubMed2006

Properties of voltage-gated Ca2+ currents measured from mouse pancreatic beta-cells in situ.

We used the single-microelectrode voltage-clamp technique to record ionic currents from pancreatic beta-cells within intact mouse islets of Langerhans at 37 degrees C, the typical preparation for studies of glucose-induced "bursting" electrical activity. Cells were impaled with intracellular microelectrodes, and voltage pulses were applied in the presence of tetraethylammonium. Under these conditions, a voltage-dependent Ca2+ current (I(Cav)), containing L-type and non-L-type components, was observed. The current measured in situ was larger than that measured in single cells with whole-cell patch clamping, particularly at membrane potentials corresponding to the action potentials of beta-cell electrical activity. The temperature dependence of I(Cav) was not sufficient to account for the difference in size of the currents recorded with the two methods. During prolonged pulses, the voltage-dependent Ca2+ current measured in situ displayed both rapid and slow components of inactivation. The rapid component was Ca2+-dependent and was inhibited by the membrane-permeable Ca2+ chelator, BAPTA-AM. The effect of BAPTA-AM on beta-cell electrical activity then demonstrated that Ca2+-dependent inactivation of I(Cav) contributes to action potential repolarization and to control of burst frequency. Our results demonstrate the utility of voltage clamping beta-cells in situ for determining the roles of ion channels in electrical activity and insulin secretion.

Animals↗

PubMed2005

Ion channel formation by Alzheimer's disease amyloid beta-peptide (Abeta40) in unilamellar liposomes is determined by anionic phospholipids.

Incorporation of Alzheimer's disease amyloid beta-proteins (AbetaPs) across natural and artificial bilayer membranes leads to the formation of cation-selective channels. To study the peptide-membrane interactions involved in channel formation, we used cation reporter dyes to measure AbetaP-induced influx of Na+, Ca2+, and K+ into liposomes formed from phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). We found that Abeta40, but not Abeta40-1 or Abeta28, caused a dose-dependent increase in the concentration of each cation in the lumen of liposomes formed from the acidic phospholipids PS and PI. The Abeta40-induced changes in cation concentration, which we attribute to ion entry through Abeta40 channels, were not observed when using liposomes formed from the neutral phospholipid PC. Using mixtures of phospholipids, the magnitude of the AbetaP40-induced ion entry increased with the acidic phospholipid content of the liposomes, with entry being observed with as little as 5% PS or PI. Thus, while negatively charged phospholipids are required for formation of cation-permeable channels by Abeta40, a small amount is sufficient to support the process. These results have implications for the mechanisms of AbetaP cytotoxicity, suggesting that even a small amount of externalized negative charge could render cells susceptible to the deleterious effects of unregulated ion influx through AbetaP channels.

Alzheimer Disease↗

PubMed2003

Loperamide mobilizes intracellular Ca2+ stores in insulin-secreting HIT-T15 cells.

1 We have investigated the effects of loperamide on intracellular Ca(2+) stores and membrane K(+) channels in insulin-secreting hamster insulinoma (HIT-T15) cells. 2 In cell-attached patch-clamp mode, loperamide (3-250 micro M) activated large single-channel currents. The loperamide-activated currents were tentatively identified as Ca(2+)-activated K(+) channel (K(Ca)) currents based on their single-channel conductance (145 pS), apparent reversal potential, and insensitivity to tolbutamide. Smaller single-channel currents with a conductance (32 pS) indicative of adenosine triphosphate-sensitive K(+) channels (K(ATP) channels) were also recorded, but were insensitive to loperamide. 3 Surprisingly, the loperamide-activated currents persisted in the absence of extracellular Ca(2+). Yet under these conditions, we still measured loperamide-induced Ca(2+) increases. These effects are dose dependent. Loperamide had no effects in the inside-out patch configuration, suggesting that loperamide does not directly activate the channels with large conductance, but does so secondarily to release of Ca(2+) from intracellular stores. 4 Carbachol (100 micro M), an agonist of muscarinic receptors, which mediates IP(3)-dependent intracellular Ca(2+) release, enhanced the effects of loperamide on K(Ca) channels. 5 Both the putative K(Ca) currents and Ca(2+) signals induced by loperamide (with '0' [Ca(2+)](o)) were abolished when the intracellular Ca(2+) stores had been emptied by pretreating the cells with either carbachol or thapsigargin, an endoplasmic reticulum Ca(2+)-ATPase inhibitor that blocks reuptake of calcium. 6 These data indicate that loperamide in insulin-secreting beta-cells evokes intracellular Ca(2+) release from IP(3)-gated stores and activates membrane currents that appear to be carried by K(Ca), rather than K(ATP) channels.

Animals↗

PubMed1996

The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo.

Formation of neuromuscular synapses requires a series of inductive interactions between growing motor axons and differentiating muscle cells, culminating in the precise juxtaposition of a highly specialized nerve terminal with a complex molecular structure on the postsynaptic muscle surface. The receptors and signaling pathways mediating these inductive interactions are not known. We have generated mice with a targeted disruption of the gene encoding MuSK, a receptor tyrosine kinase selectively localized to the postsynaptic muscle surface. Neuromuscular synapses do not form in these mice, suggesting a failure in the induction of synapse formation. Together with the results of an accompanying manuscript, our findings indicate that MuSK responds to a critical nerve-derived signal (agrin), and in turn activates signaling cascades responsible for all aspects of synapse formation, including organization of the postsynaptic membrane, synapse-specific transcription, and presynaptic differentiation.

Agrin↗

PubMed1993

Alternative forms of rat TrkC with different functional capabilities.

We have identified transcripts encoding several different forms of rat TrkC, a member of the Trk family of receptor tyrosine kinases that serves as a receptor for neurotrophin-3. Some forms of TrkC lack the intracytoplasmic kinase domain and thus resemble previously defined truncated variants of TrkB. Other forms of TrkC contain variable-sized amino acid insertions within the tyrosine kinase domain. Transcripts encoding all forms of TrkC can be detected throughout the nervous system, displaying substantial overlap as well as mutually exclusive distribution patterns with transcripts for TrkB. Strikingly, only transcripts encoding the truncated forms of TrkB and TrkC are found in astrocytes, peripheral nerve, and nonneural tissues. Finally, forms of TrkC containing insertions within the kinase domain retain their ability to autophosphorylate in response to neurotrophin-3, but cannot mediate proliferation in fibroblasts or neuronal differentiation in PC12 cells.

Amino Acid Sequence↗