RESUMO
Distinguishing between allostery and competition among modulating ligands is challenging for large target molecules. Out of practical necessity, inferences are often drawn from in vitro assays on target fragments, but such inferences may belie actual mechanisms. One key example of such ambiguity concerns calcium-binding proteins (CaBPs) that tune signaling molecules regulated by calmodulin (CaM). As CaBPs resemble CaM, CaBPs are believed to competitively replace CaM on targets. Yet, brain CaM expression far surpasses that of CaBPs, raising questions as to whether CaBPs can exert appreciable biological actions. Here, we devise a live-cell, holomolecule approach that reveals an allosteric mechanism for calcium channels whose CaM-mediated inactivation is eliminated by CaBP4. Our strategy is to covalently link CaM and/or CaBP to holochannels, enabling live-cell fluorescence resonance energy transfer assays to resolve a cyclical allosteric binding scheme for CaM and CaBP4 to channels, thus explaining how trace CaBPs prevail. This approach may apply generally for discerning allostery in live cells.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Regulação Alostérica , Ligação Competitiva , Calmodulina/metabolismo , Humanos , Ligação Proteica , Transdução de SinaisRESUMO
In the intact vertebrate central nervous system, the quantal nature of synaptic transmission is difficult to measure because the postsynaptic sites may be distributed along a tortuous dendritic tree that cannot be readily clamped spatially to a uniform potential. Titrating the intact brain's extracellular concentration of calcium ions is also challenging because of its strong buffering mechanisms. In this study, using a whole brain with eye attached preparation, quantal neurotransmission was examined in the turtle brainstem in vitro, by recording from accessory optic system neurons that receive direct input from visually responsive retinal ganglion cells. Unitary EPSPs, evoked by microstimulation of a single ganglion cell, were measured during whole cell current-clamp recordings. In this preparation, the neurons exhibit direction-selectivity, despite the hypoxic conditions. Bath application of cadmium to reduce calcium influx also reduced evoked EPSP amplitudes to that of the spontaneous synaptic events. Statistical analyses indicated that these evoked response amplitudes could be well fitted to a Poisson distribution for most brainstem neurons. Therefore, the spontaneous miniature excitatory synaptic events of approximately 1 mV, as also observed during spike blockade of the retina [Kogo, N., Ariel, M., 1997. Membrane properties and monosynaptic retinal excitation of neurons in the turtle accessory optic system. Journal of Neurophysiology 78, 614-627], are likely responses to the neurotransmitter of single vesicles release by retinal axon terminals.