Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis - PubMed (original) (raw)

Dyngo compound 4a inhibits

SVE

in synaptosomes and neurons. A–C)

SVE

was examined by quantifying uptake of

FM4

‐64 in synaptosomes stimulated with 40

mM KCl

. Dose–response curves and

IC50

values are shown for 4a (A), 6a (B) and dynasore (C). D–F)

SVE

in cultured

CGNs

. D) To further examine

SVE

inhibition by 4a,

CGNs

were loaded and unloaded with

FM1

‐43 using the protocol displayed. In both

S1

and

S2

load phases, dye was loaded into retrieving synaptic vesicles with 800 action potentials (80 Hz for 10 seconds). Unloading was stimulated by two sequential 30‐second stimuli using 50

mM KCl

. The extent of

SV

turnover was estimated from the total amount of dye unloading at

S1

(

ΔS1

) and

S2

(

ΔS2

). Where indicated, cultures were preincubated with 30 μM 4a for 15 min prior to and during either

S2

loading (Endo) or unloading (Exo). E) Cumulative histograms display the ratio of

ΔS2

to

ΔS1

across a population of single synapses. Black circles show untreated control data (Ctrl). Dyngo compound 4a was either applied during the loading phase, quantifying the effect of 4a on

SVE

(open circles), or during the unloading phase, which quantifies the effect on exocytosis (gray circles). F) The mean

ΔS2

/

ΔS1

response (±

SEM

) is displayed for control cultures (black bar,

n

= 270 nerve terminals) and for cultures where 4a was present in either the

S2

unload (gray bar,

n

= 163, exocytosis) or

S2

load (open bar,

n

= 270, endocytosis). Dyngo compound 4a had no significant effect on exocytosis, but significantly inhibited endocytosis, one‐way

ANOVA

, **p < 0.01, ***p < 0.001. G and H) The effect of 4a on whole‐cell membrane capacitance was investigated at the Calyx of Held with 0.3

mM

4a in the puffing pipette. G) A sample trace shows membrane capacitance on control (black) and 4a‐treated samples (red). H) Collated data of normalized capacitance measurement from control and 4a‐treated neurons (

n

= 6). Dyngo compound 4a‐treated samples showed no inhibition in exocytosis but a dramatic reduction in endocytosis. I) Rate of membrane retrieval (paired

t

‐test, p = 0.048). All data except (E) and (G) are means ± SEM. J) Ca2+‐dependent exocytosis from synaptosomes was measured after 30‐min incubation in 1%

DMSO

(control) or 20 μM 4a. To examine whether an activity‐dependent decrease in glutamate release was apparent with 4a treatment, the average control values were subtracted from each data point, thus representing the control sample as a relative change in glutamate release of zero. Dyngo compound 4a had no effect for the first 45–50 seconds, and subsequently caused a reduction in stimulated exocytosis that increased with stimulation time, suggesting activity‐dependent depletion in the pool of releasable synaptic vesicles in synaptosomes. Data are mean from two experiments ± range.