Rest: extracellular potential 0mV, intracellular -70mV, difference 70mV Pulse: extracellular -40mV, intracellular -70mV, difference 30mV Advantages: no capacitance – very fast rise time. Disadvantages: level of depolarization not known. Maximal depolarization = I x seal resistance. Calibration by action potential, shunt 20-25%. electrode NT muscle membrane RS

Ravin et al

N=2000 Responses=718 m=718/2000=0.36

Ex + Ca 2+ Q; 4Q + VL Ca 2+ - Hypothesis Predictions Amount of release depends strongly on [Ca 2+ ] i,o. Time course of release should depend on Ca 2+ entry and removal.

1.Entry and removal of Ca 2+ should be extremely rapid 2.Yet, residual Ca 2+ should remain for facilitation, F max =16

Depolarization-Induced Release Calcium-Induced Release Datyner & Gage Schneggenburger & Neher, 2000 Hochner et al. Bollmann & Sakmann, 2000, 2005

K. R. Delaney, R. S. Zucker, and D. W. Tank. J. Neuroscience, 1989 Ravin et al., 1999 Intra-terminal Ca 2+ measurements

Then, Why does release stop?

The answer! Ca 2+ - microdomains Ca 2+ - microdomains Ca 2+ - microdomains Ca 2+ -

Low affinity Ca 2+ sensor Time course insensitive But, sacrifice residual Ca 2+ for facilitation Conclusions:

Aharon et al

Ravin et al. (1999) found high affinity calcium sensors with Kd around 5-10  M. And indeed ! ! ! ! ! ! ! We return to square one and ask what stops evoked release especially after a train of impulses when [Ca 2+ ] i accumulates to levels of a few micromolares Schneggeburger and Neher (2000, “ Nature ” ) and Bollmann, Sakmann and Borst (2000, “ Science ” ) “ A rise in [Ca 2+ ] i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle pool in <0.5 millisecond. A comparison with action potential-evoked release suggested that a brief increase of [Ca 2+ ] i to ~10 micromolar would be sufficient to reproduce the physiological release pattern. ” Thus …

Ca-Voltage hypothesis

Thus, IF this hypothesis is correct, there should be a voltage- dependent molecule, a voltage sensor, which controls initiation and termination of fast transmitter release.

Required Properties of such a Molecule 1.Voltage Sensitive 2.Affect Time-Course of Release 3.Interact Rapidly with Release Proteins 4.Universal for all Fast Synapses

The First Step Hypothesis Enzymatic Reactions - A B C P Transmitter release P transmitter initiation fusion, pore formation stimulus - Inhibitory autoreceptors + Docking priming Voltage dependent?

InhibitoryAUTORECEPTORS  control voltage dependent initiation and termination of release  a fast process  involves formation and breakdown of protein- protein interactions  rest neurotransmitter concentration  mediate feedback inhibition  a slow process  via G-protein  high neurotransmitter concentration

Linial et al. Voltage-dependent interaction of M 2 receptor with exocytotic machinery Experimental results that provide basis for the mechanism of control of neurotransmitter release by membrane potential and autoreceptors: Rat brain synaptosomes A B

Ilouz et al Experimental results that provide basis for the mechanism of control of neurotransmitter release by membrane potential and autoreceptors Agonist binding to the receptor is required for interaction with Ex

C Experimental results that provide basis for the mechanism of control of neurotransmitter release by membrane potential and autoreceptors Voltage-dependent binding of ACh to the M 2 receptor Ilouz et al. 1999

rep RLRL Ex RLRL RLRL dep RHRH RHRH RHRH Ex (SR) Resting Potential Depolarization + Ca 2+ Q; 4Q + VL Ex Var System under TONIC BLOCK Depolarization relieves tonic block (initiation) (termination) Faster than calcium removal Slower than calcium entry

Does the GPCR affect the time course of release ? The case of the neuromuscular junction

Expected result – The time-course of release should be prolonged. a -2 RHRH RHRH RHRH Ex (SR) TERMINATIONTERMINATION What if upon repolarization re-binding of transmitter to R H is retarded? (Ex remains free longer time)

Methoctramine prolongs the time-course of ACh release Slutsky et al. 2001

dep rep RLRL Ex (ER) RLRL Ex RLRL dep rep A a -2 RHRH RHRH RHRH Ex (SR) TERMINATIONTERMINATION INITIATIONINITIATION What if R is not functional? Ex is continuously free

Then, Now Ca 2+ is the only limiting factor. The System becomes a “Ca 2+ hypothesis system” and the time course of release should depend on [Ca 2+ ] kinetics. Since there is no tonic block the rate of spontaneous release should increase. Ex + Ca 2+ Q; 4Q + VL Const.

In M 2 -KO mice the rate of spontaneous release is 3-4 times higher than in WT mice

The synaptic delay histograms of WT mice are insensitive to treatments known to affect Ca 2+ influx wt mice

The synaptic delay histograms from M 2 -KO mice are sensitive to treatments known to affect Ca 2+ influx M 2 -KO mice

The synaptic delay histograms from M 2 -KO mice, but not WT mice, are sensitive to repetitive stimulation which increases [Ca 2+ ] i M 2 -KO micewt mice

In M 2 -KO mice release starts sooner and stops later than in WT mice wt mice (n = 11) M 2 -KO mice (n = 13) ms Number of releases (Normalized to peak)

Summary & Conclusions 1)Ca 2+ entry, during and after the action potential, is faster than the relief of the tonic block. 2)After the action potential, Ca 2+ removal is slower than the reinstatement of the tonic block. Thus, Ca 2+ is not the limiting factor for initiation and termination of release.

3. When the GPCR is not functional Ca 2+ determines release kinetics. 4. Termination of release is achieved by rebinding of transmitter to the GPCR and consequent reinstatement of the tonic block.

1.The release machinery is under tonic block imposed by the inhibitory presynaptic autoreceptor M 2 R. 2.The tonic block is relieved by depolarizing pulses. The stronger the depolarization the higher the relief. 3.The autoreceptor mechanism is involved in the initiation and termination of evoked release. 4.Only when the autoreceptor mechanism is neutralized release follows predictions from the Ca 2+ -hypothesis.

1)Dual safety factor for release. 2)Low mepp frequency. (Sufficient to keep the tonic block). 3)Enables modulation of amount of release without changing time course of release (e.g. high frequency stimulation changes time course of release). 4)This is important for neuronal net – computation, tight oscillations. 5)Enables long term facilitation and yet constant time course of release.

B Ex + Ca 2+ Q; 4Q + VL Parnas & Parnas TINS 2007 dep rep RLRL Ex (ER) RLRL Ex RLRL dep rep A a -2 RHRH RHRH RHRH Ex (SR) TERMINATIONTERMINATION INITIATIONINITIATION

G-protein coupled receptors

Muscarinic receptor Metabotropic glutamate receptor GPCRs do not posses an obvious voltage sensor Voltage gated channel G

Using a “functional receptor system” to measure m2R activation 1 32

Full DR curves obtained at –60mV and +40mV revealed voltage sensitivity V (mV)Kd H (nM)Kd L (nM)R H (%)R L (%)

The binding of 3 H-ACh to the m2R is voltage dependent

Recording of charge movement associated currents (“gating currents”) 5 ms 25 nA m2R expressed oocyte Water injected oocyte Stefani E. and Bezanilla F, Methods Enzymol., 1998

Does the charge movement correlate with the affinity changes? Kd W =R H (v)Kd H +R L (V)Kd L

ACh G IP 3 Ca 2+ Cl- Channel ER PLC Shares a similar binding site with the m2R Couples to a different G-protein (G q ) Measuring m1R- mediated Ca 2+ dependent chloride currents m1R

Full DR curves obtained at –60mV and +40mV revealed voltage sensitivity V (mV)Kd H (nM)Kd L (nM)R H (%)R L (%)

Gating currents in M 1 R 5 ms 25 nA 2 ms 10 nA 2 ms 10 nA on off m1R  m2R

M 1 RECEPTOR GqGq M 2 RECEPTOR G o/i The loop that interacts with the G-protein couples the voltage sensor to the agonist binding affinity

M 1 RECEPTOR GqGq G o/i M 1 RECEPTOR/ i3 M 2 Higher affinity at depolarization Higher affinity at resting potential mV +40 mV [ACh] (nM) Response (Normalized)

M 2 RECEPTOR G o/i Higher affinity at resting potential GqGq M 2 RECEPTOR/ i3 M 1 Higher affinity at depolarization

G-protein VS (?) A G-protein A depolarization G-protein VS (?) A The loop that interacts with the G-protein couples the VS to agonist binding affinity VS 3 rd loop Agonist binding affinity VS ? (couples between the VS and the agonist binding affinity) Agonist binding affinity

M 2 RECEPTOR RIYRETENRAR ELAAL QGSETPG HISRASKSRI KKDKK EPVANQD M 1 RECEPTOR M 2 RECEPTOR Is this motif also involved in voltage sensing?

10.00 ms 50.0 nA M 2 RECEPTOR – ELAAL mutant Is this motif also involved in voltage sensing?

Outside Inside V T VS G protein

Outside Inside V T VS G protein

How General is the phenomenon of voltage sensitivity in GPCRs?

m2R – feedback inhibition of ACh release GABA B receptor – feedback inhibition of GABA release mGluR3 – feedback inhibition of glutamate release α 2 - adrenergic receptor – feedback inhibition of noradrenaline release

m1R – enhances ACh release mGluR1 – implicated in LTD