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Interpretation of presynaptic phenotypes of synaptic plasticity in terms of a two-step priming process
Full text linkStudies on synaptic proteins involved in neurotransmitter release often aim at distinguishing between their roles in vesicle priming (the docking of synaptic vesicles to the plasma membrane and the assembly of a release machinery) as opposed to the process of vesicle fusion. This has traditionally been done by estimating two parameters, the size of the pool of fusion-competent vesicles (the readily releasable pool, RRP) and the probability that such vesicles are released by an action potential, with the aim of determining how these parameters are affected by molecular perturbations. Here, it is argued that the assumption of a homogeneous RRP may be too simplistic and may blur the distinction between vesicle priming and fusion. Rather, considering priming as a dynamic and reversible multistep process allows alternative interpretations of mutagenesis-induced changes in synaptic transmission and suggests mechanisms for variability in synaptic strength and short-term plasticity among synapses, as well as for interactions between short- and long-term plasticity. In many cases, assigned roles of proteins or causes for observed phenotypes are shifted from fusion- to priming-related when considering multistep priming. Activity-dependent enhancement of priming is an essential element in this alternative view and its variation among synapse types can explain why some synapses show depression and others show facilitation at low to intermediate stimulation frequencies. Multistep priming also suggests a mechanism for frequency invariance of steady-state release, which can be observed in some synapses involved in sensory processing
How frequent are correlated changes in families of protein sequences?
No full textA loss-of-function point mutation in a protein is often rescued by an additional mutation that compensates for the original physical change. According to one hypothesis, such compensation would be most effective in maintaining a structural motif if the two mutated residues were spatial neighbors. If this hypothesis were correct, one would expect that many such compensatory mutations have occurred during evolution and that present-day protein families show some degree of correlation in the occurrence of amino acid residues at positions whose side chains are in contact. Here, a statistical theory is presented which allows evaluation of correlations in a family of aligned protein sequences by assigning a scalar metric (such as charge or side-chain volume) to each type of amino acid and calculating correlation coefficients of these quantities at different positions. For the family of myoglobins it is found that there is a high correlation between fluctuations in neighboring charges. The correlation is close to what would be expected for total conservation of local charge. For the metric side-chain volume, on the other hand, no correlation could be found
The influence of intracellular calcium concentration on degranulation of dialysed mast cells from rat peritoneum
No full text1. Mast cells, isolated from rat peritoneum, were studied under tight-seal, whole-cell recording conditions. Membrane conductance, membrane capacitance and the concentration of free intracellular Ca2+, [Ca2+]i, were measured simultaneously. 2. [Ca2+]i could be accurately buffered to values between 0 and 1.5 microM only if relatively high concentrations of calcium buffers (in the millimolar range) were added to the pipette filling solution against which the cytoplasm was dialysed. At lower buffer concentrations [Ca2+]i was markedly increased by hyperpolarizing the membrane. 3. When added to the pipette, guanosine-3-thio-triphosphate (GTP-gamma-S), a nonhydrolysable analogue of guanosine triphosphate, stimulated a 3.3-fold increase in membrane capacitance, which is indicative of mast cell degranulation (Fernandez, Neher & Gomperts, 1984). 4. In weakly buffered cells, GTP-gamma-S also induced a transient increase in [Ca2+]i which, usually, preceded degranulation. Calcium buffers at 1-5 mM concentration suppressed this transient. 5. High [Ca2+]i alone did not induce degranulation. However, it markedly accelerated GTP-gamma-S-induced degranulation. When [Ca2+]i was buffered to zero, an appreciable fraction of cells degranulated in response to GTP-gamma-S, but very slowly, and only after a long lag phase. 6. Transient increases in [Ca2+]i, evoked either by GTP-gamma-S, or by voltage changes, did not elicit capacitance changes during the lag phase, but accelerated the GTP-gamma-S-induced degranulation response at later times. 7. Internally applied inositol 1,4,5-trisphosphate (IP3) also induced transient increases in [Ca2+]i which did not lead to secretion in the absence of GTP-gamma-S. 8. It is concluded that an increase in [Ca2+]i is neither necessary nor sufficient for secretion from dialysed mast cells. [Ca2+]i, however, acts synergistically with other stimuli to promote secretion. It is the more efficient the more time the other stimulus had been allowed for priming the cell
Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.
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