1. Cellular Neuroscience (207) Ian Parker Lecture # 6 - Second messenger and Ca 2+ 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. Discrimination between ionotropic and metabotropic responses using patch-clamp recording Wall of patch-pipette Ligand applied in bathing solution cannot get under rim of pipette Ionotropic receptor would not activate, because ligand (neurotransmitter) cannot get to binding site on channel trapped within patch. Metabotropic receptor will activate channel within patch, because second messenger can diffuse within cytosol

4. 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 long after agonist is removed, owing to time taken for second messenger to be degraded 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.

5. 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) Neurotransmitter Metabotropic R Ionotropic 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).

6. Second messengers serve many functions as well as directly controlling membrane ion channels 1.Direct action on ion channels. e.g. G-protein subunits on K + channel, cGMP on channels in photoreceptors. 2.Modulation of channel activity. e.g. cAMP-dependent kinase acting via phosphoryation of channel protein. 3.Indirect control of channel activity via second messenger cascade. e.g. G-protein phospholipase IP3 Ca2+ opening of Ca2+-dependent K+ channel. 4.Modulation of enzyme activity 5.Regulation of gene transcription

7. Ca 2+ 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  M) 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

8. Calcium is the ONLY link between electrical activity of a neuron (or any other cell) and the end response of the cell: e.g. Ca 2+ influx through voltage gated channels in presynaptic terminal evokes transmitter release Ca 2+ liberation from SR triggers muscle contraction (skeletal or cardiac muscle) Ca 2+ influx through ligand-gated channels involved in LTP Unlike other ions (Na +, K +, Cl - ) it is the CHEMICAL signal carried by Ca 2+ that is important, not the electrical charge of the ion.

9. Sources of Ca 2+ ; and Ca 2+ 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 Ca 2+ 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 Ca 2+ entering through plasma membrane voltage-gated channels ER membrane- Ryanodine receptors; opening triggered by cytosolic Ca 2+ IP 3 receptors; opening requires both IP 3 and cytosolic Ca 2+

10. Only a few % of the Ca 2+ ions in the cytosol are ‘free’ Most bind to stationary Ca buffers (proteins), which slow their diffusion Ca 2+ Diffusion coefficient for free Ca 2+ in water is ~ 200  m 2 s -1, in cytosol it is about 10  m 2 s -1

11. Spatial and temporal aspects of Ca 2+ signaling Because Ca 2+ 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, Ca 2+ ions can propagate as a wave throughout a cell: e.g. to communicate signals to nucleus Sustained Ca 2+ 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. Ca 2+ Release Dynamics Ca 2+ Channel Cell Membrane Pump ER IP3 Cytosol IP 3 R Pump [Ca] Local >10  M

14. cytoplasm Ca 2+ IP 3 + IP 3 receptor Global Ca 2+ signaling Ca 2+ waves in a whole cell [Ca 2+ ]cyt

15. Global Ca 2+ signaling + - cytoplasm Ca 2+ IP 3 + IP 3 receptor + - [Ca 2+ ]cyt Ca 2+ waves in a whole cell

16. Ca 2+ -induced Ca 2+ release propagates Ca 2+ waves y x Cytosol z Cytosol space ER membrane ER store ER Cytosol Membrane

17. 10 M Local & global Ca 2+ signals

18. ‘local’ Ca 2+ signal ‘global’ Ca 2+ signal Model of local & global IP 3 - evoked Ca 2+ signals

19. IP 3 -dependent Ca 2+ signals are ordered hierarchically cellular molecular local Ca 2+ IP 3 stochasticperiodic