RIM genes differentially contribute to organizing presynaptic release sites - PubMed (original) (raw)

Comparative Study

. 2012 Jul 17;109(29):11830-5.

doi: 10.1073/pnas.1209318109. Epub 2012 Jul 2.

Affiliations

Comparative Study

RIM genes differentially contribute to organizing presynaptic release sites

Pascal S Kaeser et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Tight coupling of Ca(2+) channels to the presynaptic active zone is critical for fast synchronous neurotransmitter release. RIMs are multidomain proteins that tether Ca(2+) channels to active zones, dock and prime synaptic vesicles for release, and mediate presynaptic plasticity. Here, we use conditional knockout mice targeting all RIM isoforms expressed by the Rims1 and Rims2 genes to examine the contributions and mechanism of action of different RIMs in neurotransmitter release. We show that acute single deletions of each Rims gene decreased release and impaired vesicle priming but did not alter the extracellular Ca(2+)-responsiveness of release (which for Rims gene mutants is a measure of presynaptic Ca(2+) influx). Moreover, single deletions did not affect the synchronization of release (which depends on the close proximity of Ca(2+) channels to release sites). In contrast, deletion of both Rims genes severely impaired the Ca(2+) responsiveness and synchronization of release. RIM proteins may act on Ca(2+) channels in two modes: They tether Ca(2+) channels to active zones, and they directly modulate Ca(2+)-channel inactivation. The first mechanism is essential for localizing presynaptic Ca(2+) influx to nerve terminals, but the role of the second mechanism remains unknown. Strikingly, we find that although the RIM2 C(2)B domain by itself significantly decreased Ca(2+)-channel inactivation in transfected HEK293 cells, it did not rescue any aspect of the RIM knockout phenotype in cultured neurons. Thus, RIMs primarily act in release as physical Ca(2+)-channel tethers and not as Ca(2+)-channel modulators. Different RIM proteins compensate for each other in recruiting Ca(2+) channels to active zones, but contribute independently and incrementally to vesicle priming.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Comparative analysis of the effects of single and double RIM1α/β and RIM2α/β/γ deletions on the amplitude and Ca2+ dependence of neurotransmitter release at inhibitory synapses. Experiments in this and the following figures (except Fig. 4) were performed in cultured hippocampal neurons from conditional floxed RIM1αβf/f, RIM2αβγf/f, and/or double RIM1αβf/f/RIM2αβγf/f mice that were infected with lentiviruses expressing either active cre-recombinase (cre) or catalytically inactive, mutated cre-recombinase (control). (A) Domain structures of RIM proteins encoded by the Rims1 and Rims2 genes (Zn, N-terminal zinc finger domain, flanked by α-helical coils; S, serine corresponding to phosphorylatable serine413 in RIM1α; PDZ, central PDZ domain; C2A and C2B, central and C-terminal C2 domains, respectively; PxxP, proline-rich sequence). (B) Sample traces of IPSCs elicited by focal stimulation in neurons lacking either RIM1α/β or RIM2α/β/γ alone or together at the indicated concentrations of extracellular Ca2+ ([Ca2+]ex). Each RIM-deficient class of neurons is associated with a separate independent control. (C) Summary graphs of absolute (Upper) and normalized IPSC amplitudes (Lower; normalized to the response at 10 mM [Ca2+]ex). The lines correspond to fits of the data to a Hill function (24). (D) Summary graphs of the EC50 (Left; the [Ca2+]ex that elicits 50% of the maximal response) and of the apparent Ca2+ cooperativity (Right), determined by fitting each single experiment shown in B to a Hill function. Data shown are means ± SEMs, statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001) was determined by one-way ANOVA (C) or Student's t test (D). RIM1αβf/f: control, n = 9 cells in 3 independent batches of cultures; cre, n = 10/3; RIM2αβγf/f: control, n = 7/3; cre, n = 7/3; RIM1αβf/fxRIM2αβγf/f: control, n = 9/3; cre, n = 9/3.

Fig. 2.

Fig. 2.

Kinetic analysis of IPSCs in RIM-deficient neurons. Release was measured in response to single action potentials in cultured RIM1αβf/f, RIM2αβγf/f, and RIM1αβf/f/RIM2αβγf/f neurons expressing inactive (control) or active cre-recombinase (cre). (A) Sample traces of the initial phase of isolated evoked IPSCs in floxed RIM1αβf/f, RIM2αβγf/f, and RIM1αβf/f/RIM2αβγf/f neurons expressing inactive (control) or active cre recombinase (cre). Each trace shows five consecutive sweeps from the same neuron; note the different y scale for the different conditions. (B and C) Analyses of 20–80% rise time (B) and of the SD of the 20–80% rise time (C) as a measure of synchrony in control and RIM-deficient neurons. Data shown are means ± SEMs, statistical significance (*P < 0.05; **P < 0.01) was determined by Student’s t test (D). RIM1αβf/f: control, n = 15 cells in 3 independent batches of cultures; cre, n = 15/3; RIM2αβγf/f: control, n = 12/3; cre, n = 14/3; RIM1αβf/fxRIM2αβγf/f: control, n = 10/3; cre, n = 11/3.

Fig. 3.

Fig. 3.

Comparative analysis of the effects of single and double RIM1α/β and RIM2α/β/γ deletions on release induced by 10-Hz stimulus trains. Release was measured in response to trains with 2–20 action potentials that were elicited by focal stimulation at 10 Hz in RIM1αβf/f, RIM2αβγf/f, and RIM1αβf/f/RIM2αβγf/f neurons expressing inactive (control) or active cre recombinase (cre). (A) Sample traces of IPSCs in response to a train of 20 stimuli. (B) Summary graphs of the synaptic charge transfer evoked by the first action potential in trains of 2, 5, 10, and 20 action potentials. (C) Summary graphs of the total charge transfer during each entire stimulus train. (D) Plots of the ratios of the evoked amplitudes of the last to the first response in each train. (E) Summary graphs of delayed release, measured as the charge transfer starting 100 ms after the last action potential was applied in the train. Data are shown as means ± SEMs. Statistical significance in (B_–_E): *P < 0.05; **P < 0.01; ***P < 0.001 calculated by one-way ANOVA (RIM1αβf/f: control, n = 6 cells in 3 independent batches of cultures; cre, n = 6/3; RIM2αβγf/f: control, n = 9/3; cre, n = 9/3; RIM1αβf/f/RIM2αβγf/f: control, n = 10/3; cre, n = 14/3).

Fig. 4.

Fig. 4.

Effects of RIM1α and RIM2γ on N-type and L-type Ca2+ channels in HEK293T cells. HEK293T cells were transfected with Ca2+-channel α-subunits (N-type, CaV2.2, A-G; L-type, CaV1.2, H-N), auxiliary β4b and α2δ subunits, and either RIM1α or RIM2γ. (A_–_C) Experimental protocol (A), sample traces (B), and summary (C) of the current-voltage relationship of N-type CaV2.2 channels in the absence or presence of RIM1α or RIM2γ (control, n = 15 cells in 4 batches of independent transfections; RIM1α, n = 10/4; RIM2γ, n = 13/3). (D and E) Experimental protocol (D) and summary plot (E) of the activation curve of N-type CaV2.2 channels in the absence or presence of RIM1α or RIM2γ (control, n = 12/4; RIM1α, n = 13/5; RIM2γ, n = 12/4). (F and G) Experimental protocol (F) and summary data (G) of the inactivation curve (control, n = 9/4; RIM1α, n = 9/5; RIM2γ, n = 9/4) of CaV2.2 channels in the absence or presence of RIM1α or RIM2γ. (H–J) Experimental protocol (H), sample traces (I), and summary (J) of the current-voltage relationship of L-type CaV1.2 channels in the absence or presence of RIM1α or RIM2γ (control, n = 11/ 3; RIM1α, n = 14/4; RIM2γ, n = 7/3). (K and L) Experimental protocol (K) and summary plot (L) of the activation curve of L-type CaV1.2 channels in the absence or presence of RIM1α or RIM2γ (control, n = 12/3; RIM1α, n = 13/3; RIM2γ, n = 12/3). (M and N) Experimental protocol (M) and summary data (N) of the inactivation curve (control, n = 7/3; RIM1α, n = 6/3; RIM2γ, n = 10/4) of CaV1.2 channels in the absence or presence of RIM1α or RIM2γ. Data shown are means ± SEMs, statistical significance: ***P < 0.001 for RIM1α and RIM2γ compared with control determined by two-way ANOVA. No other significant differences in two-way ANOVA analyses were observed.

Fig. 5.

Fig. 5.

Effect of RIM2γ on the extracellular Ca2+ dependence of neurotransmitter release and on the priming of synaptic vesicles. Release was measured in RIM1αβf/f/RIM2αβγf/f neurons expressing inactive (control) or active cre-recombinase (cre), or cre + RIM2γ expressed bicistronically from the same lentivirus as described (14, 19, 21). (A) Sample traces of single evoked IPSCs, the extracellular Ca2+-concentration ([Ca2+]ex) was varied as indicated. (B) Summary graphs of absolute IPSC amplitudes. (C) Summary graphs of normalized IPSC amplitudes normalized to the response at 10 mM [Ca2+]ex. (D) EC50 (Left; [Ca2+]ex that elicits 50% of the maximal response) and apparent Ca2+ cooperativity of release (Right), determined by fitting each single experiment shown in B to a Hill function (24). (E) Sample traces of IPSCs evoked by 0.5 M sucrose application from RIM1αβf/f/RIM2αβγf/f neurons after cre and control infection, or infection with a virus expressing cre + RIM2γ. (F) Summary graphs of the synaptic charge transfer during the initial (1–10 s) and the steady-state (15–30 s) phases of the sucrose response are shown. Data shown are means ± SEMs; statistical significance: **P < 0.01; ***P < 0.001 was determined by one-way ANOVA (B and C) or Student’s t test (D and F), with control, n = 5/2; cre, n = 5/2; cre + RIM2γ; n = 4/2 for B_–_D; and control, n = 8/3; cre, n = 10/3; cre + RIM2γ, n = 10/3 for F.

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