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Biomedical subjects

T E Fisher

Publications and source records attributed to T E Fisher.

At least 19 recordsLinked to original sources
PubMed2001

The function of Ca(2+) channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release.

By mediating the Ca(2+) influx that triggers exocytotic fusion, Ca(2+) channels play a central role in a wide range of secretory processes. Ca(2+) channels consist of a complex of protein subunits, including an alpha(1) subunit that constitutes the voltage-dependent Ca(2+)-selective membrane pore, and a group of auxiliary subunits, including beta, gamma, and alpha(2)-delta subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca(2+) channels are constituted by different combinations of alpha(1) subunits (of which 10 have been identified) and auxiliary subunits, particularly beta (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca(2+) channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca(2+) channel structure and function in exocytotic secretion. We discuss Ca(2+) channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca(2+) channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca(2+) channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.

Animals↗
PubMed2000

Intracellular Ca(2+) channel immunoreactivity in neuroendocrine axon terminals.

The concentration of neuroendocrine terminals in the neurohypophysis facilitates the identification and localization of Ca(2+) channel subtypes near neuroendocrine release sites. Immunoblots of rat neurohypophysial tissue identified the alpha(1)1.3, alpha(1)2.1, alpha(1)2.2, and alpha(1)2.3 Ca(2+) channel subunits. Immunofluorescence staining of axon terminal plasma membranes was weak, suggesting that Ca(2+) channels are dispersed. This contrasts with the highly punctate alpha(1)2.2 immunoreactivity in bovine chromaffin cells; the neurohypophysial terminals may therefore lack the specialized release zones found in those cells. Immunofluorescence and immunogold labeling identify dense core granule-like structures in the terminal cytoplasm containing multiple Ca(2+) channel types. Ca(2+) channels in internal membranes may play an important role in channel targeting and distribution in neuroendocrine cells.

Animals↗
PubMed2000

Mechanical design of proteins studied by single-molecule force spectroscopy and protein engineering.

Mechanical unfolding and refolding may regulate the molecular elasticity of modular proteins with mechanical functions. The development of the atomic force microscopy (AFM) has recently enabled the dynamic measurement of these processes at the single-molecule level. Protein engineering techniques allow the construction of homomeric polyproteins for the precise analysis of the mechanical unfolding of single domains. alpha-Helical domains are mechanically compliant, whereas beta-sandwich domains, particularly those that resist unfolding with backbone hydrogen bonds between strands perpendicular to the applied force, are more stable and appear frequently in proteins subject to mechanical forces. The mechanical stability of a domain seems to be determined by its hydrogen bonding pattern and is correlated with its kinetic stability rather than its thermodynamic stability. Force spectroscopy using AFM promises to elucidate the dynamic mechanical properties of a wide variety of proteins at the single molecule level and provide an important complement to other structural and dynamic techniques (e.g., X-ray crystallography, NMR spectroscopy, patch-clamp).

Elasticity↗
PubMed2000

Stretching single molecules into novel conformations using the atomic force microscope.

A dense network of interconnected proteins and carbohydrates forms the complex mechanical scaffold of living tissues. The recently developed technique of single molecule force spectroscopy using the atomic force microscope (AFM) has enabled a detailed analysis of the force-induced conformations of these molecules and the determinants of their mechanical stability. These studies provide some of the basic knowledge required to understand the mechanical interactions that define all biological organisms.

Animals↗
PubMed1999

The micro-mechanics of single molecules studied with atomic force microscopy.

The atomic force microscope (AFM) in its force-measuring mode is capable of effecting displacements on an angstrom scale (10 A = 1 nm) and measuring forces of a few piconewtons. Recent experiments have applied AFM techniques to study the mechanical properties of single biological polymers. These properties contribute to the function of many proteins exposed to mechanical strain, including components of the extracellular matrix (ECM). The force-bearing proteins of the ECM typically contain multiple tandem repeats of independently folded domains, a common feature of proteins with structural and mechanical roles. Polysaccharide moieties of adhesion glycoproteins such as the selectins are also subject to strain. Force-induced extension of both types of molecules with the AFM results in conformational changes that could contribute to their mechanical function. The force-extension curve for amylose exhibits a transition in elasticity caused by the conversion of its glucopyranose rings from the chair to the boat conformation. Extension of multi-domain proteins causes sequential unraveling of domains, resulting in a force-extension curve displaying a saw tooth pattern of peaks. The engineering of multimeric proteins consisting of repeats of identical domains has allowed detailed analysis of the mechanical properties of single protein domains. Repetitive extension and relaxation has enabled direct measurement of rates of domain unfolding and refolding. The combination of site-directed mutagenesis with AFM can be used to elucidate the amino acid sequences that determine mechanical stability. The AFM thus offers a novel way to explore the mechanical functions of proteins and will be a useful tool for studying the micro-mechanics of exocytosis.

Animals↗
PubMed1999

Pulsed laser imaging of Ca(2+) influx in a neuroendocrine terminal.

The surge of Ca(2+) that triggers vesicle fusion is shaped by the distribution of Ca(2+) channels and the physical relationship between those channels and the exocytotic apparatus. Although channels and the release apparatus are thought to be tightly associated at fast synapses, the arrangement at neuroendocrine cells is less clear. The distribution of Ca(2+) influx near release sites is difficult to determine because of spatial and temporal limitations on Ca(2+) imaging techniques. We now present spatially resolved images of Ca(2+) influx into rat neuroendocrine terminals on a millisecond time scale. Images of voltage-dependent Ca(2+) influx into neurohypophysial terminals were captured after excitation of Ca(2+)-sensitive dyes with pulses of laser light lasting a fraction of a microsecond. Submembranous Ca(2+) increases were detected during the first millisecond of an evoked Ca(2+) tail current. Steep gradients of Ca(2+) were evident, with concentrations near the membrane reaching above 1 microM during a 30 msec depolarization. Ca(2+) influx appeared evenly distributed, even when diffusion was restricted with an exogenous Ca(2+) chelator. During longer depolarizations, mean and peak Ca(2+) concentrations reached an asymptote in parallel, suggesting that Ca(2+) binding proteins near the membrane rapidly buffer Ca(2+) and do not become saturated during prolonged influx. These data support the hypothesis that exocytosis is activated in these terminals by the summation of influx through multiple, randomly spaced Ca(2+) channels.

Animals↗
PubMed1999

The study of protein mechanics with the atomic force microscope.

The unfolding and folding of single protein molecules can be studied with an atomic force microscope (AFM). Many proteins with mechanical functions contain multiple, individually folded domains with similar structures. Protein engineering techniques have enabled the construction and expression of recombinant proteins that contain multiple copies of identical domains. Thus, the AFM in combination with protein engineering has enabled the kinetic analysis of the force-induced unfolding and refolding of individual domains as well as the study of the determinants of mechanical stability.

Entropy↗
PubMed1998

Density of transient K+ current influences excitability in acutely isolated vasopressin and oxytocin neurones of rat hypothalamus.

1. The transient outward K+ current (ITO) was studied using whole-cell recording in immunocytochemically identified oxytocin (OT; n = 23) and vasopressin (VP; n = 67) magnocellular neurosecretory cells (MNCs) acutely isolated from the supraoptic nucleus of adult rats. 2. The peak density of ITO during steps to -10 mV was 26 % smaller in OT-MNCs (355 +/- 23 pA pF-1; mean +/- s.e. m.; n = 18) than in VP-MNCs (478 +/- 17 pA pF-1; n = 52). No differences were observed in the voltage dependence of activation or inactivation. 3. Kinetic analysis revealed two components of ITO inactivation in both OT-MNCs (tau1 = 9.2 +/- 0.4 ms and tau2 = 41.2 +/- 1.6 ms; n = 18) and VP-MNCs (tau1 = 12.4 +/- 0.4 ms and tau2 = 37.1 +/- 1.2 ms; n = 52). Although the density of the rapid component (tau1) was not different (275 +/- 13 versus 265 +/- 16 pA pF-1, respectively), the slow component (tau2) was markedly smaller in OT-MNCs (183 +/- 19 versus 331 +/- 16 pA pF-1 in VP-MNCs). 4. In unidentified MNCs, 0.5 mM 4-aminopyridine reduced ITO amplitude by 29% and decreased the latency to spike discharge by about 70% during depolarization from -70 mV. Latency to discharge from potentials less negative than -60 mV, where ITO is inactivated, was unaffected. 5. Comparison of latency to spike discharge in identified cells showed that OT-MNCs achieve spike threshold twice as fast as VP-MNCs when depolarized from -70 mV. The lower density of ITO in OT-MNCs, therefore, accelerates the rate at which excitation can occur in response to depolarizing stimuli and may facilitate the occurrence of higher frequency discharges in OT-MNCs during physiological activation.

Animals↗
PubMed1998

Properties of the transient K+ current in acutely isolated supraoptic neurons from adult rat.

The transient outward current (ITO) in magnocellular neurosecretory cells was studied using whole cell patch clamp recordings made from supraoptic neurons acutely isolated from the adult rat. In the presence of tetrodotoxin, depolarizing steps applied from a negative holding potential evoked a rapidly activating and inactivating outward current followed by a slowly activating and sustained outward current. The ITO was unaffected by tetraethylammonium (TEA), but was blocked by 4-aminopyridine. The ITO evoked by depolarization from negative potentials in the presence of 40 mM TEA was not different from that revealed by digital subtraction of current traces recorded with and without a negative conditioning prepulse in control solutions. Tail current analysis during perfusion of media containing different external [K+] indicated that ITO is selective for K+ ions. Exposure to Ca(2+)-free solutions containing divalent inorganic cations such as Cd2+ and Ni2+ could cause shifts in voltage-dependency and, thereby, reduce the amplitude of ITO recorded at fixed submaximal potentials, The maximal ITO achievable under these conditions, however, was reduced compared to control. Moreover, the ITO was also reduced by application of organic Ca2+ channel antagonists such as nifedipine and omega-conotoxin GVIA, indicating that a component of the ITO is somehow dependent on Ca2+ influx.

4-Aminopyridine↗
PubMed1996

Identification and characterization of a Ca(2+)-sensitive nonspecific cation channel underlying prolonged repetitive firing in Aplysia neurons.

The afterdischarge of Aplysia bag cell neurons has served as a model system for the study of phosphorylation-mediated changes in neuronal excitability. The nature of the depolarization generating the afterdischarge, however, has remained unclear. We now have found that venom from Conus textile triggers a similar prolonged discharge, and we have identified a slow inward current and corresponding channel, the activation of which seems to contribute to the onset of the discharge. The slow inward current is voltage-dependent and Ca(2+)-sensitive, reverses at potentials slightly positive to O mV, exhibits a selectivity of K approximately equal to Na >> Tris > N-methyl-D-glucamine (NMDG), and is blocked by high concentrations of tetrodotoxin. Comparison of these features with those observed in channel recordings provides evidence that a Ca(2+)-sensitive, nonspecific cation channel is responsible for a slow inward current that regulates spontaneous repetitive firing and suggests that modulation of the cation channel underlies prolonged changes in neuronal response properties.

Animals↗
PubMed1996

Calcium-channel subtypes in the somata and axon terminals of magnocellular neurosecretory cells.

To understand the specific functions of Ca(2+)-channel types it is necessary to know how they are distributed within neurons. The unique structure of the magnocellular neurosecretory cells of the rat supraoptic nucleus has made it possible to obtain whole-cell recordings from individual somata and axon terminals acutely isolated from adult rats. Characterization of elicited Ca2+ currents in these cells has demonstrated that certain types are segregated in somata or axon terminals, and that current types defined pharmacologically can display different kinetic properties in the two loci. Observed biophysical properties correlate with functional requirements in the two compartments and have implications for the roles of specific Ca(2+)-channel subtypes.

Animals↗
PubMed1995

Distinct omega-agatoxin-sensitive calcium currents in somata and axon terminals of rat supraoptic neurones.

1. Voltage-dependent calcium currents were measured at room temperature using whole-cell patch clamp in acutely isolated somata and axon terminals of the magnocellular neurosecretory cells (MNCs) from the rat supraoptic nucleus. 2. Administration of omega-agatoxin IVA (omega-Aga IVA) blocked a high-threshold non-inactivating current. This current has an IC50 for omega-Aga IVA of 3 nM; no other types of currents were blocked at doses of up to 500 nM. 3. In the axon terminals omega-Aga IVA blocked a high-threshold current that inactivates markedly (tau approximately 448 ms), and has a much lower sensitivity to the toxin, with an IC50 of 270 nM. Unlike the somatic current, the effect of omega-Aga IVA in the terminals is largely prevented by omega-conotoxin GVIA (omega-CgTX). 4. These data suggest that MNC somata express a single type of omega-Aga IVA-sensitive calcium current similar to the P-type calcium current described in other cells. However, the omega-Aga IVA-sensitive current in axon terminals differs from both the P-type and the recently identified Q-type current in that it is also sensitive to omega-CgTX. The distinct biophysical properties of the currents in somata and axon terminals may have important physiological implications.

Animals↗
PubMed1995

Voltage-gated calcium currents in the magnocellular neurosecretory cells of the rat supraoptic nucleus.

1. Whole-cell patch-clamp techniques were used to analyse voltage-dependent calcium currents in acutely isolated somata of magnocellular neurosecretory cells (MNCs) from the supraoptic nucleus of the hypothalamus of adult rats. Currents were characterized on the basis of their rates of inactivation and their sensitivity to a series of calcium channel blocking agents. 2. Curve fitting analysis of series of long lasting depolarizing voltage steps from a holding potential of -80 mV revealed three current components with different voltage dependences and rates of inactivation (n = 36). These include a low threshold (-60 mV), rapidly inactivating (tau = 42 +/- 3 ms at -10 mV) component, a high threshold (-30 mV), slowly inactivating (tau = 1790 +/- 70 ms) component and a component with an intermediate threshold (-50 mV) and rate of inactivation (tau = 187 +/- 15 ms). There is also a non-inactivating portion of evoked calcium current with a threshold of -50 mV. 3. Based on its voltage dependence, rate of inactivation, greater sensitivity to the divalent cation nickel than to cadmium and insensitivity to omega-conotoxin GVIA (omega-CgTX), the low threshold current appears to be a T-type calcium current. The rate of inactivation, voltage dependence, and sensitivity to omega-CgTX of the slowly inactivating component suggests that it is an N-type current. The characteristics of the intermediate component do not correspond to any identified calcium current type. 4. Portions of the non-inactivating calcium current are sensitive to nifedipine (23 +/- 2% of the total non-inactivating current at -10 mV; n = 10), suggesting the presence of L-type currents, omega-agatoxin-IVA (omega-Aga-IVA; 20 +/- 6% of total; n = 11), suggesting the presence of P-type channels, and omega-CgTX (39 +/- 3% of total; n = 19), suggesting the presence of a non-inactivating N-type current. The non-inactivating component at low potentials (> or = -50 mV) was selectively blocked by nifedipine, suggesting the presence of a novel, low threshold L-type current. 5. We conclude that MNC soma express T-, N-, L-, and P-type calcium currents, as well as a novel low threshold nifedipine-sensitive current and an unidentified inactivating component. This complement of currents is different from that seen in the terminals of these cells, suggesting a spatial and functional segregation of calcium current types in MNCs.

Animals↗
PubMed1994

Synthesis and biological activity of ras farnesyl protein transferase inhibitors. Tetrapeptide analogs with amino methyl and carbon linkages.

Replacement of the central amino methylene linkage of C[psi CH2NH]A[psi CH2NH]AX tetrapeptide inhibitors with carbon tethers led to compounds with potency in the nanomolar range. Some of the more potent olefinic compounds inhibit Ras processing in intact v-ras transformed NIH 3T3 cells with IC50 values in the 0.1 to 1 microM range, and inhibit selectively the anchorage-independent growth of H-ras transformed Rat1 cells at 10 microM.

3T3 Cells↗
PubMed1994

Transient changes in intracellular calcium associated with a prolonged increase in excitability in neurons of Aplysia californica.

1. Transient stimulation of an afferent input to the bag cell neurons of Aplysia californica triggers a 30-min period of spontaneous firing termed the afterdischarge. Measurement of free calcium ion concentrations using calcium-sensitive electrodes revealed a biphasic pattern of elevation of intracellular calcium levels during the afterdischarge. Basal calcium levels at the soma were found to rise rapidly during afferent stimulation and then to decline before the onset of spontaneous firing. This early peak in intracellular calcium was followed by a slower, transient elevation of calcium levels during the period of rapid firing that occurs in the first few minutes of afterdischarge. Stimulation of clusters of bag cell neurons in a calcium-free external medium failed to trigger afterdischarge and produced no changes in basal intracellular calcium levels. 2. When calcium ions in the external medium were replaced by barium ions, stimulation of clusters of bag cell neurons triggered afterdischarges that were characterized by long-duration action potentials. Intracellular calcium levels during these afterdischarges rose slowly over the first few minutes of spontaneous firing. Because calcium-sensitive microelectrodes do not respond to barium ions, these data suggest that stimulation of afterdischarge triggers calcium release from an intracellular compartment. 3. During afterdischarges in barium-containing external media, each broadened action potential produced a discrete transient elevation of intracellular calcium levels. A similar effect was observed in isolated bag cell neurons in primary culture when action potentials were stimulated by depolarizing current pulses in a barium-containing medium. These data suggest that, under these conditions, individual action potentials trigger the release of intracellular calcium from some intracellular pool.

Afferent Pathways↗