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M E Diaz

Publications and source records attributed to M E Diaz.

7 recordsLinked to original sources
PubMed2004

Stability and instability of regulation of intracellular calcium.

[Ca2+]i is used as a signal in many tissues. In this review we discuss the mechanisms that regulate [Ca2+]i and, importantly, what determines their stability. Brief mention is made of the effects of feedback gain and delays on stability. The control of cytoplasmic Ca concentration is shown to be generally stable as Ca pumping is essentially an instantaneous function of [Ca2+]i. In contrast, regulation of the Ca content of intracellular stores may be less stable. One example of this is instability in the control of sarcoplasmic reticulum (SR) Ca content in cardiac muscle. An increase of SR Ca content increases the systolic Ca transient amplitude. This in turn decreases Ca influx into the cell and increases efflux, thereby restoring SR Ca to control levels. This feedback system has an inherent delay and is potentially unstable if the gain is increased beyond a certain level. This instability produces Ca transients of alternating amplitude and may contribute to the clinical syndrome of pulsus alternans.

Adaptation, Physiological↗
PubMed1999

beta subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunit compositions in lethargic mouse brain.

Neuronal voltage-dependent Ca2+ channels are heteromultimers of alpha1, beta, and alpha2delta subunits, and any one of five alpha1 subunits (alpha1A-E) may associate with one of four beta subunits (beta1-4). The specific alpha1-beta combination assembled determines single-channel properties, while variation in the proportion of each combination contributes to the functional diversity of neurons. The mouse mutant lethargic (lh) exhibits severe neurological defects due to a mutation that deletes the alpha1 subunit interaction domain of the beta4 subunit. Since beta subunits regulate critical alpha1 subunit properties in heterologous expression systems, loss of beta4 in lethargic could dramatically alter channel localization and behavior unless beta1-3 subunits can be used as substitutes in vivo. Here we demonstrate increased steady-state associations of alpha1A and alpha1B with the remaining beta1-3 subunits, without significant changes in beta1-3 mRNA abundance. The immunolocalization of alpha1A and alpha1B protein in lethargic brain is indistinguishable from wild-type by light microscopy. Furthermore, the measurement of large-amplitude P-type currents in dissociated lethargic Purkinje neurons indicates that these alpha1A-containing channels retain regulation by beta subunits. We conclude that several properties of alpha1A and alpha1B proteins are not uniquely regulated by beta4 in vivo and may be rescued by beta1-3 subunit reshuffling. The complex neurological manifestation of the lethargic mutation therefore emerges from loss of beta4 coupled with the widespread pairing of surrogate beta subunits with multiple Ca2+ channel subtypes. The existence of beta subunit reshuffling demonstrates that molecular plasticity of Ca2+ channel assembly, a normal feature of early brain development, is retained in the mature brain.

Animals↗
PubMed1998

Mutation in AP-3 delta in the mocha mouse links endosomal transport to storage deficiency in platelets, melanosomes, and synaptic vesicles.

The mouse mutant mocha, a model for the Hermansky-Pudlak storage pool deficiency syndrome, is characterized by defective platelets, coat and eye color dilution, lysosomal abnormalities, inner ear degeneration, and neurological deficits. Here, we show that mocha is a null allele of the delta subunit of the adaptor-like protein complex AP-3, which is associated with coated vesicles budding from the trans-Golgi network, and that AP-3 is missing in mocha tissues. In mocha brain, the ZnT-3 transporter is reduced, resulting in a lack of zinc-associated Timm historeactivity in hippocampal mossy fibers. Our results demonstrate that the AP-3 complex is responsible for cargo selection to lysosome-related organelles such as melanosomes and platelet dense granules as well as to neurotransmitter vesicles.

Adaptor Protein Complex 3↗
PubMed1998

Role of potassium channels in amyloid-induced cell death.

Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid beta-peptide (A beta) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to A beta. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K+ current with delayed rectifier characteristics. Increases of 64% (+/-19; p < 0.02) and 44% (+/-12; p < 0.02) in K+ current density were noted 6-12 and 12-18 h following the addition of A beta to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 +/- 4 and 39 +/- 4% of SN56 cells were dead after 48- and 96-h exposures to A beta, respectively. Perfusion of SN56 cells with 10-20 mM TEA blocked 71 +/- 6 to 92 +/- 2% of the outward currents, widened action potentials, elevated [Ca2+]i, and inhibited 89 +/- 14 and 68 +/- 14% of the A beta toxicity. High [K+]o, which depolarizes cell membranes and increases [Ca2+]i, also protected SN56 cells from A beta toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K+ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, A beta was toxic to a dopaminergic cell line (MES23.5) that expressed a K+ current with delayed rectifier characteristics; K+ current density was not altered by A beta and MES23.5 cells were not protected by TEA from A beta toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K+ currents was resistant to the toxicity of A beta. These data suggest that a K+ channel with delayed rectifier characteristics may play an important role in A beta-mediated toxicity for septal cholinergic cells.

Amyloid beta-Peptides↗
PubMed1997

Modulation of septal cell activity by extracellular zinc.

Zinc released from axon terminals in the brain can interact with multiple membrane channels and receptors. However, the specific effects of these Zn(2+)-dependent interactions on physiological processes remains unclear. Because Zn(2+)-containing axon terminals are abundant in the septal region, we selected a septal cell line (SN56) to study the effects of Zn2+ on cell activity. Voltage-clamp recordings showed well-developed voltage-dependent Na+, Ca2+ and K+ currents. Micromolar concentrations of Zn2+ partially blocked Na+ and Ca2+ currents without affecting K+ currents. Current-clamp recordings showed that SN56 cells fire spontaneous and evoked action potentials. While most (> or = 83%) Na+ and Ca2+ currents were blocked with 1 microM tetrodotoxin (TTX) and 2 mM Co2+, action potentials persisted after either 1 microM TTX or 2 mM Co2+ application. In contrast, concentrations of Zn2+ (50-300 microM) that induced incomplete blockade (< or = 50%) of either Ca2+ and Na+ currents abolished action potential generation. These data show that simultaneous and partial blockade of Ca2+ and Na+ channels by Zn2+ inhibit SN56 cell activity. Because septal outputs extensively modulate the excitability of cortical and subcortical brain regions, Zn2+ inhibition of action potential generation in septal neurons could play an important physiological role in regulating brain activity.

Action Potentials↗
PubMed1997

Developmental expression of chick cortical GABA(A) receptor alpha1 subunits in vivo and in vitro.

In order to examine the expression of the GABA(A) receptor alpha1 subunit during chick cortical development in vivo and in vitro, we have utilized a polyclonal antibody (RP4) directed against an alpha1(331-381) fusion protein. This antibody exhibits a high titer for precipitation of [3H]flunitrazepam binding sites in chick cortical extracts, no significant cross-reactivity with GABA(A) receptor beta2- or beta4-subunit fusion proteins, and a robust reaction with a single 51-kDa polypeptide on immunoblots of cortical membranes. This indicates monospecificity of the RP4 antiserum for the GABA(A) receptor alpha1 subunit. The alpha1-subunit antibody also showed strong immunocytochemical reactions with neurons in the embryonic mediodorsal cortex and Purkinje cells of the chick cerebellum. The ontogeny of the alpha1 subunit in chick cortex and in derived neuronal cultures was examined by quantitative Western blotting. The level of the alpha1 polypeptide increased from day 2 to day 6 in culture, acquiring 50% of the maximum expression at day 4. Expression of the cortical GABA(A) receptor alpha1 subunit increased in vivo from embryonic day 8 (E8) to day 7 post-hatching, reaching 50% of adult levels at E16. Levels of the corresponding alpha1-subunit mRNA, analyzed from E8 to E20 by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR), showed a corresponding incline. These findings correlated well with previous developmental studies of GABA(A) receptor ligand binding sites both in vivo and in vitro. The parallel increase of the alpha1 subunit transcript and polypeptide with [3H]flunitrazepam binding sites suggests that this subunit may be an important component of GABA(A) receptors early in cortical ontogeny. This was investigated further by quantitative immunoprecipitation. At saturation, the RP4 antiserum consistently precipitated 50-65% of the central [3H]flunitrazepam binding sites in the developing cortex from E12 through P7, despite a 5-fold increase in the binding level. The data suggest that during cortical development the fraction of GABA(A) receptors containing alpha1 subunits remains relatively constant. Furthermore, the alpha1 polypeptide appears to be a major component of GABA(A) receptor oligomers at all stages of cortical maturation.

Animals↗