BS2550 - Neuronal and Cellular Signalling Lecture 13 Calcium There are 4 mechanisms for regulating calcium: - Cell membrane. - Excretion systems. - Calcium channels. - Intracellular organelles. Calcium signalling is based upon calcium ion concentration gradients. These gradients are created by energy dependent calcium transport across cellular membranes. Sodium/calcium exchanges: - Direction of movement of ions is governed by membrane potential. - This is important for the removal of calcium. Physiological stimulation opens plasmalemmal or intracellular calcium channels thus creating calcium fluxes affecting the concentration of calcium ions in various intracellular compartments. NMDA receptor is a glutamate receptor that is key for regulating calcium ion levels. It is permeable to sodium, potassium, and calcium. A loss of magnesium block during depolarisation allows for ion flow and a large amount of calcium enters the cell. Calmodulin: - A ubiquitous, small, acidic protein containing four EF hand motifs, each of which binds calcium. - It binds calcium and then targets proteins to modify their activity. - Composed of two globular domains each containing two calcium binding sites. - Once calcium binds, protein confirmation changes causing hydrophobic patches on the protein that can be exposed which mediate interaction. Calmodulin can bind several kinases called Calmodulin kinases based on the substrate specificity. - One substrate > dedicated calcium/CaM-dependent kinases. - Brad substrate specificity > multifunctional kinases. Calcium signals in localised domains: - Calcium diffuses slowly in cells since it binds negative charges.
Calcium ion concentration increases from the resting level to approximately 1uM. the increased calcium binds to intracellular substrates leading to modulation of cell function. The speed of calcium ion concentration is derived from: - Rapid but transient calcium release from intracellular calcium stores. - Slow calcium influx from extracellular space. Calcium influx through voltage gated calcium channels from the extracellular space is particular important for regulation of calcium in the synapse. Synaptotagmin is a calcium binding protein that needs to be bound to the SNARE complex for pore opening to occur. Calcium channels are closely associated with the SNARE complex. Allosteric changes happen due to its opening. These are suggested to be part of the rearrangement that allows the pore to open. Calcium Dyes Quantification using fluorescent dye indicators - dyes contain aromatic rings that chelate calcium resulting in changes in their absorption and fluorescence properties upon binding calcium. There are two types of calcium dyes: - Non-ratiometric - on binding, the calcium due increases n fluorescence intensity. - Ratiometric - dye binding calcium causes an emission spectral shift or excitation spectral shift. Calcium sensing using dyes: - Fura-2 is a ratiometric due allowing visualisation of calcium levels. - A membrane permanent calcium chelator that functions as a dye-molecule. - Derived from EGTA with 4 COOH groups to bind calcium. - Calcium binding alters fluorescence. - This can be used for quantitative measuring of calcium. Intracellular calcium concentrations can be displayed using ratio values for wavelengths of 340 over 380. - Rationing minimizes unwanted effects like uneven dye loading. Calcium influx