Neuroscience Fundamentals 112C Ian Parker

1. Neuroscience Fundamentals 112CIan Parker Biophysics of intracellular neuronal signaling Second messenger and Ca2+ signaling

2. Ionotropic and metabotropic receptors Ionotropic (direct coupled) receptor. Receptor site and channel are part of the same molecular conmplex Metabotropic (indirectly coupled) receptor. Ligand receptor and channel are distinct entities, expressed by different genes, and physically separated in the membrane. Functional link between them is via a diffusible ‘second messenger’ (or chain of several second messengers). (The extracellular ligand is the ‘first messenger’)

3. Functional characteristics of ionotropic vs. metabotropic responses. MetabotropicIonotropic 1. Amplification and diversity of responses A single receptor can generate many second messenger molecules, which can activate many channels; and activate more than one type of channel. No amplification. Receptor site is part of same molecular complex as the channel, so can control only that channel. Also, a given receptor type will control only a single channel type. 2.Slow responses Channel activation depends on complex pathway. Generation of messenger causes initial delay. Then response can persist Fast responses Everything is built into one molecular complex, so channel can open within microseconds of agonist application. Similarly, channel can close within milliseconds as agonist dissociates from receptor site. long after agonist is removed, owing to time taken for second messenger to be degraded

4. 3. Function depends on many molecules Complex system, requires energy for generation and recycling of second messengers. Will not function in excised patches Function depends on only a single molecule No energy needed. Channel continues to function in excised patch. There are many different metabotropic receptors (more receptors than there are second messengers) NeurotransmitterMetabotropic RIonotropic R Ionotropic receptors mediate fast through-put of information in the nervous system (e.g. when brake lights go on on truck ahead of you!). Metabotropic receptors are concerned with slower modulation of activity (e.g. wakefulness).

5. Second messengers serve many functions as well as directly controlling membrane ion channels • Direct action on ion channels. e.g. G-protein subunits on K+ channel, cGMP on channels in photoreceptors. • Modulation of channel activity. e.g. cAMP-dependent kinase acting via phosphoryation of channel protein. • Indirect control of channel activity via second messenger cascade. e.g. G-protein phospholipase IP3 Ca2+ opening of Ca2+-dependent K+ channel. • Modulation of enzyme activity • Regulation of gene transcription

6. Ca2+ signaling Resting [Ca 2+ ] in cytosol maintained at ~ 50 nM by actions of pumps & exchangers. So, only a little Ca entering the cytosol will give big (a few mM) increase in concentration. Cytosolic Ca can…… Activate membrane ion channels Regulate enzyme activity Modulate protein function Regulate gene expression Kill cells! (necrotic & apoptotic death) It has functions in virtually all cells of the body. Christened a ‘life and death’ messenger

7. Calcium is the ONLYlink betweenelectrical activity of a neuron (or any other cell) and the end response of the cell:e.g. Ca2+ influx through voltage gated channels in presynaptic terminal evokes transmitter release Ca2+ liberation from SR triggers muscle contraction (skeletal or cardiac muscle) Ca2+ influx through ligand-gated channels involved in LTPUnlike other ions (Na+, K+, Cl-) it is the CHEMICAL signal carried by Ca2+ that is important, not the electrical charge of the ion.

8. Sources of Ca2+; and Ca2+ permeable channels Extracellular fluid (~ 2 mM) Plasma membrane channels- Voltage-gated channels (N, P, Q, L-type) Ligand-gated channels: e.g. several neurotransmitter- activated channels have appreciable Ca2+ permeability (nicotinic ACh, NMDA) Store-operated channels: open in response to depletion of e.r calcium stores Endoplasmic/sarcoplasmic reticulum (~ 1mM) SR membrane (skeletal muscle)- Ryanodine receptors; opening coupled to voltage sensors in plasma membrane SR membrane (cardiac muscle)- Ryanodine receptors; opening triggered by Ca2+ entering through plasma membrane voltage-gated channels ER membrane- Ryanodine receptors; opening triggered by cytosolic Ca2+ IP3 receptors; opening requires both IP3 and cytosolic Ca2+

9. DiffusionThe only way calcium ions can transmit information from one place in a cell to another. • A particle (ion, molecule, organelle, whatever…) undergoing diffusion follows a ‘random walk’ [run web movie] • The mean distance L it will have moved (in 3-dimensions) from its starting point after time t is given by L = sqrt (6Dt) ; where D = diffusion coefficient (um2 sec-1) • So, mean distance increases as square root of time • E.g. a molecule with D = 17 um2 sec-1 will travel 100 nm in 0.1 ms; 1 mm in 10 ms; 10 mm in 1 s; 100 mm in ~2 min. Compare ‘chemical’ and electrical signaling in neurons…

10. Only a few % of the Ca2+ ions in the cytosol are ‘free’ Most bind to stationary Ca buffers (proteins), which slow their diffusion Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Diffusion coefficient for free Ca2+ in water is ~ 200 mm2 s-1, in cytosol it is about 10 mm2 s-1

11. Spatial and temporal aspects of Ca2+ signaling Because Ca2+ diffusion in the cell is hindered by immobile buffers, transient Ca 2+ elevations can be highly localized, and specifically regulate only nearby targets – e.g. neurotransmitter release. Or, Ca2+ ions can propagate as a wave throughout a cell: e.g. to communicate signals to nucleus Sustained Ca2+ elevations kill cells (e.g. glutamate neurotoxicity). So Ca waves are generated periodically. Frequency of repetitive waves encodes stimulus strength.

12. ‘Digital’ vs. ‘Analog’ encoding of information Weak stimulus Strong stimulus Strength encoded by frequency or pattern of all-or none signals Strength encoded in a continuously-graded manner by signal amplitude

13. IP3/Ca2+ signaling pathway IP3 Cytosol Ca2+ Channel [Ca]Local>10mM Pump IP3R Cell Membrane ER Pump

14. Ca2+ IP3 + cytoplasm IP3 receptor Global Ca2+ signaling Ca2+ waves in a whole cell [Ca2+]cyt

15. Ca2+ IP3 + + - cytoplasm IP3 receptor Global Ca2+ signaling Ca2+ waves in a whole cell [Ca2+]cyt - +

16. Cytosol z y Cytosol space x ER membrane ER store Ca2+-induced Ca2+ release propagates Ca2+ waves Cytosol Membrane ER

17. Local & global Ca2+ signals 10 mM

18. Model of local & global IP3 - evoked Ca2+ signals ‘local’ Ca2+ signal ‘global’ Ca2+ signal

19. IP3-dependent Ca2+ signals are ordered hierarchically Ca2+ IP3 local cellular molecular stochastic periodic