Ionic dependence of Ca channel modulation by syntaxin 1A

Ionic dependence of Ca2ⴙ channel modulation by syntaxin 1A Ofer Wiser, Roy Cohen, and Daphne Atlas* Department of Biological Chemistry Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel Communicated by Lutz Birnbaumer, National Institutes of Health, Research Triangle Park, NC, January 10, 2002 (received for review September 16, 2001)

Alteration of the kinetic properties of voltage-gated Ca2ⴙ channels, Cav1.2 (Lc-type), Cav2.2 (N type), and Cav2.3 (R type), by syntaxin 1A (Syn1A) and synaptotagmin could modulate exocytosis. We tested how switching divalent charge carriers from Ca2ⴙ to Sr2ⴙ and Ba2ⴙ affected Syn1A and synaptotagmin modulation of Ca2ⴙchannel activation. Syn1A accelerated Cav1.2 activation if Ca2ⴙ was the charge carrier; and by substituting for Ba2ⴙ, Syn1A slowed Cav1.2 activation. Syn1A also significantly accelerated Cav2.3 activation in Ca2ⴙ and marginally in Ba2ⴙ. Synaptotagmin, on the other hand, increased the rate of activation of Cav2.3 and Cav2.2 in all permeating ions tested. The Syn1A-channel interaction, unlike the synaptotagmin-channel interaction, proved significantly more sensitive to the type of permeating ion. It is well established that exocytosis is affected by switching the charge carriers. Based on the present results, we suggest that the channel-Syn1A interaction could respond to the conformational changes induced within the channel during membrane depolarization and divalent ion binding. These changes could partially account for the charge specificity of synaptic transmission as well as for the fast signaling between the Ca2ⴙ source and the fusion apparatus of channel-associatedvesicles (CAV). Furthermore, propagation of conformational changes induced by the divalent ions appear to affect the concerted interaction of the channel with the fusion兾docking machinery upstream to free Ca2ⴙ buildup and兾or binding to a cytosolic Ca2ⴙ sensor. These results raise the intriguing possibility that the channel is the Ca2ⴙ sensor in the process of fast neurotransmitter release. exocytosis 兩 synaptotagmin 兩 Ca2⫹ sensor 兩 strontium 兩 transmitter release

alcium (Ca2⫹) entry through voltage-gated Ca2⫹ channels is essential for the initiation of excitation-secretion coupling in neuronal and neuroendocrine cells. Other divalent cations such as Ba2⫹ and Sr2⫹ are able to move through Ca2⫹ channels and mimic Ca2⫹ action (1–5). The divalent charge carriers affect transmitter release at the neuromuscular junction (1, 5–7) and the squid giant synapse (3) as well as the neuronal (8–13) and neuroendocrine cells (14). Substitutions of the extracellular charge carriers bring about a complex series of effects on channel behavior that have been categorized into three mechanisms (15). The electrostatic actions of the divalent ions affect the membrane field (16–18); they also interact with sites at the channel pore (19–21) and induce conformational changes after binding at specific regulatory sites, influencing gating (22, 23). Therefore, differences observed on the extent and kinetics of secretion by various divalents cannot be attributed entirely to Ca2⫹-channel inactivation or binding to an intracellular Ca2⫹ sensor but could involve a change in conformation induced by binding of the divalent ion to the channel and passage through the pore. Recent studies have shown that voltage-gated Ca2⫹ channels are coupled physically and functionally to the exocytotic proteins syntaxin 1A (Syn1A), SNAP-25, and synaptotagmin (24–37). Monitoring Ca2⫹-channel interactions with synaptic proteins in the Xenopus oocyte expression system led to the proposal that voltage-gated Ca2⫹-channel and exocytotic machinery are cou-

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pled intimately (38). These studies demonstrated the assembly of Cav1.2 or Cav2.2 with Syn1A, SNAP-25, and synaptotagmin into the excitosome complex (30, 34). The close proximity generated between the Ca2⫹ source and the synaptic proteins is expected to promote a higher efficiency of transmitter release. Indeed, such structural design might be responsible for the highly efficient insulin secretion (500–600 granules per sec) observed at rather low Ca2⫹ influx through a relatively few number of Ca2⫹ channels (⬍500) in islet cells (39). Previously we have shown that Syn1A modifies Cav1.2 and Cav2.2 activation (30, 37, 38). Here we demonstrate that different charge carriers variously alter this modification, and we propose that the conformational change induced in the channel during ion binding and permeation is responsible for it. This signaling pathway could be relevant for the time course of the onset of evoked secretion where the Ca2⫹ channel contacts with the SNARE protein directly, in turn gaining control of the fusion process. Conformational changes that are induced by the instant binding of Ca2⫹ to the channel appear to be propagated to the ensuing docking兾fusion machinery. These results suggest that the Ca2⫹ channel is the Ca2⫹ sensor of transmitter release, operating upstream to an intracellular Ca2⫹ sensor. Methods cDNA Constructs. ␣ 1 1.2 (dN60-del1773; X15539), rat ␤ 2A (m80545), and ␣12.3 subunit (L27745; ref. 34) were obtained from N. Qin and L. Birnbaumer (National Institutes of Health, Triangle Research Park, NC), and ␣2兾 ␦ rabbit skeletal (M86621) was obtained from A. Schwartz (University of Cincinnati, Cincinnati). ␣11.2 from T. P. Snutch (University of British Columbia, BC) was inserted into pGEM-He (37). SytI was from M. L. Bennett (University of California, Berkeley, CA). The in vitro transcription kit was from Stratagene. Electrophysiological Assays. Stage V and VI oocytes were removed surgically from female Xenopus laevis and injected with cRNA mixtures encoding Ca2⫹ channel subunits. One day later, cRNA of Syn1A, synaptotagmin, or water (control) was injected (28, 30). Whole-cell currents were recorded by applying a standard two-electrode voltage clamp as described (30). In initial experiments, inward currents in solutions containing Mn2⫹, Ca2⫹, Sr2⫹, and Ba2⫹ were recorded sequentially from a single oocyte. Because of current run-down, an alternative protocol was applied in which groups of oocytes (n ⫽ 80) that expressed the channel either alone or with synaptic proteins were subdivided for recording in various ions (n ⫽ 15–20). The pulse durations for activation were: Cav1.2, 140 msec given in 10-sec intervals; Cav2.2, 1.4 sec in 30-sec intervals; and Cav2.3, 200 msec in 10-sec intervals. Current traces were leaksubtracted online by the CLAMPEX7 software, and channel activation rates were analyzed by applying a monoexponential fit of Abbreviation: Syn1A, syntaxin 1A. *To whom reprint requests should be addressed. E-mail: [email protected]. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

www.pnas.org兾cgi兾doi兾10.1073兾pnas.052017299

the CLAMPFIT 6 software (Axon Instruments, Foster City, CA) to the current traces at the relevant ranges. Fit ⫽ A exp [t兾␶act] ⫹ B, where A ⫽ current amplitude, ␶act ⫽ time constant of activation, and t ⫽ time to peak. Activation was determined from the beginning of the trace just after the capacitative transient to the peakcurrent region. The capacitative transient that lasted for 1–2.5 msec was subtracted online. Data Presentation and Statistical Analysis. Peak current and ␶act

were analyzed by CLAMPFIT 6 and transferred to an EXCEL worksheet (Microsoft), data were averaged for each group of oocytes, and SE was determined. The data were presented as mean ⫾ SE. Statistical significance relative to the control group was calculated by Student’s t test. Comparison of statistical significance between the various groups in each experiment was determined with the one-way ANOVA test using ORIGIN 5 software (Microcal, Amherst, MA). Results Charge Carriers Affect Syn1A–Cav1.2 Interaction. We have shown previously that Syn1A slows Cav1.2 activation and promotes current inhibition (30). Here we compared the effect of charge carriers on channel modulation by Syn1A. Cav1.2 currents were generated in oocytes injected with cRNA encoding ␣11.2 (2 ng per oocyte), ␣2␦1 (1.5 ng per oocyte), and ␤2a (5 ng per oocyte). Syn1A cRNA (3 ng per oocyte) or water (control) was injected Wiser et al.

1 day later, and currents were recorded on day 6 or 7. The kinetic properties of Cav1.2 were determined in external solutions containing 5 mM Ca2⫹, 5 mM Ba2⫹, 5 mM Sr2⫹, or 5 mM Mn2⫹ in 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acidinjected oocytes (Fig. 1). The effect of charge carriers on the current amplitude was monitored first with four separate solutions containing the indicated divalent ions applied consecutively to the same oocyte. Cav1.2 currents in each solution were evoked by 1,600 msec steps from a holding potential of ⫺80 mV to ⫹15 mV. Inward currents were affected differentially, and the amplitude of peak currents of a single representative oocyte was significantly larger in Ba2⫹ than those triggered in Sr2⫹ or Ca2⫹ (Ba2⫹ ⬎ Sr2⫹ ⬎ Ca2⫹ ⬎⬎ Mn2⫹; Fig. 1, Top Left). When the channel was coexpressed with Syn1A, Cav1.2 currents elicited by a 140-msec step to ⫹15 mV showed a similar, although not identical, order for the peak amplitude, Ba2⫹ ⬎ Sr2⫹ ⬎ Ca2⫹ ⱖ Mn2⫹ (Fig. 1, Bottom Left). Likewise, normalized traces showed how the rates were altered after switching the external solution from Mn2⫹ to Ca2⫹, to Sr2⫹, and to Ba2⫹ (Fig. 1, Top Right). Most likely, the conformational changes induced at the channel by ion binding and permeation are reflected in channel modulation by Syn1A. These were demonstrated by replotting normalized currents of the channel with and without Syn1A for each divalent ion (Fig. 1, Bottom Right). As shown previously, Syn1A decreased Cav1.2 activation in 5 mM Ba2⫹ (Fig. 1; refs. 30 and 37). Conversely, it increased the activation rate in Ca2⫹ and Sr2⫹ but not in Mn2⫹ (Fig. 1, Bottom Right). We analyzed the contribution of the fastinactivating component to ␶act (Fig. 1, Middle). A twoexponential fit showed ␶2 ⫽ 308 ⫾ 2 msec and ␶1 ⫽ 4,984 ⫾ 117 msec in 5 mM Ba2⫹. Stimulation of ␶inact from 250 to 350 msec in 10-msec increments and t in 0.1-msec increments ␶act was PNAS 兩 March 19, 2002 兩 vol. 99 兩 no. 6 兩 3969

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Fig. 1. Charge carriers modify Syn1A–Cav1.2 activation. Oocytes were injected with cRNA encoding ␣11.2 (2 ng), ␣2.␦1 (1.5 ng), ␤2a (5 ng), and 1 day later with Syn1A (3 ng) or water (control). (Top Left) Superposition of representative online leak-subtracted current traces measured in divalent ion solutions as indicated with voltage protocols diagramed at the top. (Inset) Two exponential fits of ␶inact. (Bottom Left) Superposition of representative online leak-subtracted Cav1.2 current traces with Syn1A in various ions elicited in response to 140 msec to ⫹10 mV. (Top Right) The first 30 msec of the response is shown. (Bottom Right) The same representative currents as in Lower Left, normalized and replotted for a better display of the effect of Syn1A on Cav1.2 activation. *, oocytes coexpressing Cav1.2 and Syn1A.

Fig. 2. Syn1A modifies Cav1.2 current amplitude in various divalent charge carriers. Cav1.2 peak currents were evoked by 140-msec test pulses to ⫹10 mV either alone (Top) or with Syn1A (Middle) in different divalent solutions. Currents were averaged from groups of 15 oocytes. The data are shown as mean ⫾ SE. The percent of current inhibition is plotted for each divalent ion as indicated (Bottom).

identical experimental conditions (see above). The time constant of activation (␶act) was determined at voltages ranging from ⫺10 to ⫹40 mV (Fig. 3 A and B). As demonstrated, the rate was fastest in Ba2⫹ and slower in Ca2⫹ or Sr2⫹, and the slowest rate was observed in Mn2⫹ in the order of Ba2⫹ ⬎ Sr2⫹ ⱖ Ca2⫹ ⬎ Mn2⫹ (Fig. 3A; Table 1). The voltage dependency of ␶act estimated from the slopes was similar in the four cations (Fig. 3A). When associated with Syn1A, Cav1.2 activation was altered significantly compared with that in Cav1.2 alone, especially in Ca2⫹, where the rate was faster than in the absence of Syn1A: Ca2⫹ ⬎ Sr2⫹ ⱖ Ba2⫹ ⬎⬎ Mn2⫹ in the voltage range of ⫺10 to ⫹15 mV (Fig. 3B). In addition, Cav1.2 activation in the presence of Syn1A was considerably less sensitive to voltage changes in the ⫺10 to ⫹10-mV range compared with Cav1.2 alone (see also Fig. 1 Middle). To resolve the ion effect on Syn1A–channel interaction, ␶act was replotted for each ion solution (Fig. 4). Syn1A increased ␶act of Cav1.2 in Ba2⫹ (Fig. 4A; Table 1; ref. 30). In contrast, ␶act was significantly lower when Ca2⫹ was the charge carrier, and the voltage sensitivity of activation was virtually eliminated (Fig. 4D). Syn1A caused a smaller reduction in ␶act in Sr2⫹, and a reduction in voltage sensitivity was prominent only in the ⫺10 to ⫹10-mV range (Fig. 4B). For Mn2⫹, Syn1A induced a significantly smaller change in ␶act, and the voltage dependency of activation was unaffected (Fig. 4C). In contrast to Ba2⫹, Syn1A had no apparent effect on ␶act in Ca2⫹, Sr2⫹, or Mn2⫹ at potentials higher than ⫹20 mV (Fig. 4C). The ␶act of Cav1.2 in four different external divalents was compared at ⫹10-mV test pulses, emphasizing opposite modulation in Ba2⫹ and Ca2⫹ (Fig. 4E). The unique decrease of ␶act by Syn1A in the presence of Ca2⫹ also was observed in EGTA-injected oocytes (data not shown).

Fig. 3. Charge carriers affect the Syn1A–Cav1.2 interaction. Oocytes were injected with cRNA encoding Cav1.2 subunits (Fig. 1) and 1 day later with Syn1A cRNA (3 ng) or water. Currents were evoked in response to 140-msec pulses in 5-mV increments. Online leak-subtracted currents in oocytes expressing Cav1.2 alone (A, open symbols) or with Syn1A (B, closed symbols). Each group of oocytes was placed in recording solution containing 5 mM Ba2⫹ (circles), 5 mM Ca2⫹ (squares), 5 mM Sr2⫹ (triangles), and 5 mM Mn2⫹ (diamonds). The time constant of activation (␶act) was determined by a single exponential fit of the onset of current traces. Each dot corresponds to the mean ⫾ SE (n ⫽ 10 –20).

changed from 5.28 to 5.15 msec and did not contribute significant to our ␶act measurements. Although the effects of divalent ions on the channel–Syn1A interaction were noticeable, the exhaustive perfusion applied to the oocytes was harmful, causing current run-down. To circumvent these difficulties, we monitored a large number of oocytes (n ⫽ 10–15 for each divalent solution) injected from the same cRNA batch and tested on the same day with various ions. Fig. 2 compares peak-current amplitudes of CaV1.2 in the presence of the four different divalent ions: Ba2⫹ ⬎ Sr2⫹ ⬎ Ca2⫹ ⬎ Mn2⫹ (Fig. 2 Top). The largest amplitude was observed in Ba2⫹ and the smallest in Mn2⫹. They ran as follows: Ba2⫹ ⬎ Sr2⫹ ⬎ Ca2⫹ ⬎ Mn2⫹ (Fig. 2 Top). With Syn1A, the potency order was modified to Ba2⫹ ⬎⬎ Sr2⫹ ⬎ Mn2⫹ ⱖ Ca2⫹ (Fig. 2 Middle). Hence, the Syn1A inhibitory pattern of current amplitude was strongest in Ca2⫹ and weakest in Ba2⫹ (Fig. 2 Bottom). This result led to further analysis of divalent ion effects on Cav1.2 activation in the absence and presence of Syn1A under

Synaptotagmin–Channel Interaction Is Not Modified by Divalent Ions.

Synaptotagmin has been shown previously to interact physically with domains II and III of Cav2.2, Cav2.1, and Cav1.2 (29–31, 34, 40). Functionally, synaptotagmin increased Cav2.2 activation but not that of Cav1.2. It also reversed the inhibitory effects of Syn1A on Cav2.2 and Cav1.2 (refs. 30, 34, and 36–38; Table 1) and those of SNAP-25 on Cav2.2 (36). To determine whether switching permeating ions affected synaptotagmin–Cav2.2 interaction, we measured Cav2.2 activity in oocytes coexpressed with synaptotagmin in Ba2⫹, Sr2⫹, or Ca2⫹ applied consecutively to the same oocyte (5 mM; Fig. 5). In each solution, current was evoked from

Table 1. Differential effect of Syn1A and synaptotagmin on current amplitude and activation of voltage-gated Ca2ⴙ channels relative to various divalent charge carriers ⫹Syntaxin

Control

Cav1.2 Ba2⫹ Ca2⫹ Sr2⫹ Mn2⫹ Cav2.2 Ba2⫹ Ca2⫹ Sr2⫹ Cav2.3 Ba2⫹ Ca2⫹

Peak, nA

␶act, ms

Peak, nA

␶act, ms

⫺2801 ⫾ 147 ⫺1380 ⫾ 115 ⫺1588 ⫾ 180 ⫺586 ⫾ 36

2.61 ⫾ 0.14 3.04 ⫾ 0.08 3.3 ⫾ 0.1 4.0 ⫾ 0.27

⫺624 ⫾ 74* ⫺109 ⫾ 21* ⫺250 ⫾ 35* ⫺138 ⫾ 15*

3.39 ⫾ 0.16* 2.47 ⫾ 0.06* 3.28 ⫾ 0.07 3.75 ⫾ 0.13

⫺413 ⫾ 50 ⫺128 ⫾ 31 ⫺257 ⫾ 123

200 ⫾ 6 264 ⫾ 15 201 ⫾ 13

⫺1914 ⫾ 133 ⫺2147 ⫾ 93

6.3 ⫾ 0.32 10.5 ⫾ 0.54

⫹Synaptotagmin Peak, nA

2.4 ⫾ 0.18 3.12 ⫾ 0.2 3.0 ⫾ 0.3

⫺340 ⫾ 85 ⫺151 ⫾ 29 ⫺246 ⫾ 67 ⫺1490 ⫾ 135 ⫺1715 ⫾ 92*

5.52 ⫾ 0.22 7.5 ⫾ 0.77**

␶act, ms

⫺1532 ⫾ 80 ⫺2048 ⫾ 112

164 ⫾ 14 217 ⫾ 7 165 ⫾ 23 4.2 ⫾ 0.12 8.2 ⫾ 0.00

Currents were recorded in response to depolarization steps in solutions containing 5 mM Ba2⫹, 5 mM Ca2⫹, 5 mM Sr2⫹, and 5 mM Mn2⫹. Activation time constants (␶act) were determined by a pulse to ⫹15 mV (Cav1.2 and Cav2.2) and to ⫺5 mV in Cav2.3. Oocytes were injected with 5 mM 1,2-bis(aminophenoxy)ethane-N,N,N⬘,N⬘tetraacetic acid prior to recording. Statistical significance was determined by applying two-sample Student’s t test. *, P ⬍ 0.01. **, P ⬍ 0.02. 3970 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.052017299

Wiser et al.

Fig. 4. Specific effect of Syn1A on Cav1.2 activation in various ions. The data presented in Fig. 3 was replotted to demonstrate ␶act modification by Syn1A at each divalent ion solution. The recording solution contained 5 mM Ba2⫹ (A), 5 mM Sr2⫹ (B), 5 mM Ca2⫹ (C), or 5 mM Mn2⫹ (D). Each dot corresponds to mean ⫾ SE (n ⫽ 10 –20). (E) Summary of the results in A–D: ␶act of Cav1.2 currents evoked by a test pulse to ⫹10 mV (white) and Syn1A (gray). *, Student’s t test, P ⬍ 0.01.

a holding potential of ⫺80 mV in response to a 1,400-msec test pulse to ⫹15 mV. Superposition of Cav2.2 current traces expressed with synaptotagmin in different divalents showed peak currents with a similar rank order of Ba2⫹ ⬎ Sr2⫹ ⬎ Ca2⫹ (Fig. 5 Left). Switching the permeating ions did not alter the Cav2.2– synaptotagmin interaction (Fig. 5 Middle), demonstrating a similar increase in the rate of activation (Fig. 5 Right). The synaptotagmin effect was apparent for each ion solution when ␶act collected from a large number of oocytes was plotted

Fig. 6. Modulation of Cav2.2 activation by synaptotagmin. Oocytes were injected with cRNA encoding Cav2.2 subunits and 24 h later with synaptotagmin cRNA (Œ) or water (E). Inward currents were evoked by a 1.4-sec depolarizing step from a holding potential of ⫺80 mV to various test potentials (⫺10 to ⫹40 mV) in 5-mV increments. The recording solution contained 5 mM Ba2⫹ (A), 5 mM Sr2⫹ (B), or 5 mM Ca2⫹ (C). The plotted ␶act values correspond to mean ⫾ SE of 10 –20 oocytes. (D) The ␶act of Cav2.2 at ⫹15 mV in different divalents. (E) The ␶act of Cav2.2 coexpressed with synaptotagmin normalized by ␶act of the channel alone (see D). St, synaptotagmin.

against the voltage (Fig. 6; Table 1). Synaptotagmin caused a notable increase in Cav2.2 activation at all voltages in the three divalent ions (Fig. 6 A–C). The ␶act of Cav2.2 associated with synaptotagmin was significantly higher in Ca2⫹ relative to Sr2⫹ (Fig. 6D; P ⬍ 0.001) or Ba2⫹ (Fig. 6D; P ⬍ 0.001; ref. 38). Despite a higher ␶act in Ca2⫹ (Fig. 6 C and D), there was no significant difference in the ␶act ratio of Cav2.2 whether with or without synaptotagmin in Ba2⫹ or Sr2⫹ (Fig. 6E). Thus, switching the permeating ions did not alter modulation of ␶act by synaptotagmin.

Fig. 5. Cav2.2 interaction with synaptotagmin. Oocytes were injected with the cRNA encoding Cav2.2 currents ␣12.2 (5 ng), ␣2␦1 (3 ng), and ␤2a (7 ng), and 1 day later with synaptotagmin (5 ng) or water. Inward currents were recorded sequentially in Ca2⫹, Sr2⫹, and Ba2⫹ with extensive washes between solutions. (Left) Superposition of representative online leak-subtracted current traces evoked by 1.4-sec test pulse (Upper) to ⫹10 mV. (Center) The same traces as those in Left normalized for better visualization of the sensitivity to charge carrier identity. (Right) Same traces replotted, displaying the synaptotagmin effect on ␶act of Cav2.2 in different ions as indicated.

Wiser et al.

erated by ␣12.3兾␤2A兾␣2␦1 (41) coexpressed along with Syn1A or synaptotagmin (see Fig. 8). Current traces were measured with a voltage protocol as diagramed and normalized (Fig. 7A). Current amplitudes were inhibited modestly in both Ca2⫹ (23%, Fig. 7B Left) and Ba2⫹ (20%, Fig. 7C Left; Table 1). Syn1A significantly accelerated Cav2.3 activation in Ca2⫹ during a 200-msec test pulse (⫺10 to ⫹5 mV; Fig. 7B Right), whereas in Ba2⫹, activation increased marginally at all voltages tested (Fig. 7C Right). The difference in ␶act in Ca2⫹ and Ba2⫹ at ⫺10 mV is shown in Fig. 7D. Current amplitudes of Cav2.3 were not affected by synaptotagmin in Ba2⫹ and slightly reduced (⬍20%) in Ca2⫹ (Fig. 8 A and B Left; Table 1). Cav2.3 activation was accelerated by synaptotagmin to an extent similar to its effect on Cav2.2 (Fig. 8 A and B Right; ref. 38). Activation was increased to a similar extent in Ca2⫹ at ⫺10 to 0-mV range and in Ba2⫹ at ⫺10 to ⫹5 mV. The differences in ␶act in Ca2⫹ and Ba2⫹ at ⫺5 mV are shown in Fig. 8C. Thus, switching the permeating ions from Ba2⫹ to Ca2⫹ altered modulation of ␶act by Syn1A but not synaptotagmin. Discussion Charge Carriers Affect Syn1A Interaction with Cav1.2. In this study we demonstrated that switching the permeating ions appeared to affect the voltage-gated Ca2⫹-channel interaction differentially with Syn1A. Because both Syn1A and the divalent ions shape channel kinetics, it is difficult to establish whether a conformaPNAS 兩 March 19, 2002 兩 vol. 99 兩 no. 6 兩 3971

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Switching Charge Carriers Modify the Cav2.3–Syn1A but Not Cav2.3– Synaptotagmin Interaction. Fig. 7 illustrates Cav2.3 currents gen-

Fig. 8. Ca2⫹ and Ba2⫹ effect on Cav2.3 interaction with synaptotagmin. Oocytes were injected with cRNA encoding Cav2.3 subunits (Fig. 7) and 24 h later with synaptotagmin cRNA (5 ng, Œ) or water (E). Leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three-channel subunit with and without synaptotagmin in solutions containing 5 mM Ca2⫹ (A) or 5 mM Ba2⫹ (B). The data points correspond to the mean ⫾ SE of current (n ⫽ 8 –10). Two-sample Student’s t test assuming unequal variance was applied. The plotted ␶act values correspond to mean ⫾ SE. (C) Difference in ␶act with and without synaptotagmin in Ca2⫹ and Ba2⫹ at ⫺5 mV. Fig. 7. Cav2.3 interaction with Syn1A in Ca2⫹ and Ba2⫹. Oocytes were injected with cRNA encoding ␣12.3 (5 ng)兾␣2␦1 (5 ng)兾␤2a (10 ng) and 24 h later with Syn1A (4 ng), synaptotagmin (5 ng), or water (E). (A) Superposition of online subtracted current traces in 5 mM Ca2⫹ and 5 mM Ba2⫹. Currents were evoked from a holding potential of ⫺80 mV in response to a 200-msec depolarizing step to 0 mV. (B) Leak-subtracted peak current-voltage relationship: collected data from oocytes expressing the three-channel subunit with (F) and without Syn1A (E) in solutions containing 5 mM Ca2⫹ or 5 mM Ba2⫹ (C). The data points correspond to the mean ⫾ SE of current (n ⫽ 8 –10). Twosample Student’s t test assuming unequal variance was applied. The ␶act was determined by a single exponential fit of the current traces. The plotted values correspond to mean ⫾ SE (n ⫽ 8 –10). (D) The difference in ␶act with and without Syn1A in Ca2⫹ and Ba2⫹ at ⫺5 mV.

tional change induced by the divalent ions affected the Syn1A interaction or whether Syn1A coupling with the channel is responsible for altering channel sensitivity toward the divalent ions. Cav1.2 peak current was significantly larger in Ba2⫹ than in Sr2⫹ or Ca2⫹, and this order is similar to the potency order observed in the presence of Syn1A. However, Syn1A induced a greater reduction in Ca2⫹ flux compared with other ions in the external solution. This result implies that the basic permeation properties of the ␣11.2 subunit intrinsic to the channel might alter the channel interaction with Syn1A. The voltage dependency of Cav1.2 activation in the various ions also was affected by the presence of Syn1A. When the charge carriers were switched from Ba2⫹ to Ca2⫹ or Sr2⫹, the moderate voltage dependency in the absence of Syn1A became 3972 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.052017299

more restrained with Syn1A in Ca2⫹. Syn1A induced two opposite effects on channel kinetics when the charge carriers were switched; in Ca2⫹ it increased Cav1.2 activation at the ⫺10 to ⫹20-mV range, and in Ba2⫹ activation was decreased at voltages from ⫺10 to ⫹40 mV. Similar results were obtained for the R-type channel (Cav2.3). The significant increase in the Cav2.3 activation in Ca2⫹, a channel that does not inactivate in Ca2⫹, suggests no connection to Ca2⫹ inactivation. These results demonstrate modulation of Cav2.3 by synaptic proteins, which is consistent with Cav2.3 support of fast release (42, 43). The contribution of Syn1A-induced faster activation in Ca2⫹ to the time course of release requires additional study. It is tempting to speculate that the permeating ions instigated changes in the gating of the channel (44–49), affecting its interaction with Syn1A. Syn1A is essential for membrane fusion; therefore, such changes propagated from the channel to Syn1A could modify fusion, an attractive model to explain a ‘‘stimuluscoupled’’ process of minimal latency between Ca2⫹ influx and transmitter release. In fact, such a mechanism implies a control of synaptic dynamics through Ca2⫹ binding to the channel rather than free Ca2⫹ buildup and binding to an intracellular Ca2⫹ sensor (ref. 50; see below). Synaptotagmin Interaction with Ca2ⴙ Channel Is Insensitive to Divalent Ions. Synaptotagmin accelerated the activation of Cav2.3 and

Cav2.2 (38) but not Cav1.2 (30). The increase in the neuronal channels activation was observed irrespective of the ion type.

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Correlation to Evoked Release. It has been established that the

charge carriers shape transmitter release. Our results show that the Syn1A–channel interaction is sensitive to switching the charge carrier, suggesting that this interaction denotes a molecular coupling of the channel to the release machinery. The channel could modify depolarization-evoked secretion through Syn1A, a delegate of the fusion machinery, and the modification of the stimulus-coupled event would be compatible with the divalent type. Given that the channel provides the Ca2⫹ needed for exocytosis, it acts both by controlling ion flux and directly through interaction with the synaptic proteins. Recently, we showed that fast insulin secretion from pancreatic cells was inhibited by the cytosolic II–III domain of Cav1.2, 1. Meiri, U. & Rahamimoff, R. (1971) J. Physiol. (London) 215, 709–726. 2. Mellow, A. M., Perry, B. D. & Silinsky, E. M. (1982) J. Physiol. (London) 328, 547–562. 3. Augustine, G. J. & Eckert, R. (1984) J. Physiol. (London) 346, 257–271. 4. Miledi, R. (1996) Nature (London) 212, 1233–1234. 5. Bain, A. I. & Quastel, D. M. (1992) J. Physiol. (London) 450, 63–87. 6. Miledi, R. & Slater, C. R. (1966) J. Physiol. (London) 184, 473–498. 7. Dodge, F. A., Jr. & Rahamimoff, R. (1967) J. Physiol. (London) 193, 419–432. 8. Wigstrom, H. & Swann, J. W. (1980) Brain Res. 194, 181–191. 9. Goda, Y. & Stevens, C. F. (1994) Proc. Natl. Acad. Sci. USA. 91, 12942–12946. 10. Abdul-Ghani, M. A., Valiante, T. A. & Pennefather, P. S. (1996) J. Physiol. (London) 495, 113–125. 11. Capogna, M., McKinney, R. A., O’Connor, V., Gahwiler, B. H. & Thompson, S. M. (1997) J. Neurosci. 17, 7190–7202. 12. Rumpel, E. & Behrends, J. C. (1999) J. Physiol. (London) 514, 447–458. 13. Xu-Friedman, M. A. & Regehr, W. G. (1999) Biophys. J. 76, 2029–2042. 14. Weiss, C., Sela, D. & Atlas, D. (1990) Neurosci. Lett. 119, 241–244. 15. McNaughton, N. C. & Randall, A. D. (1997) Neuropharmacology 36, 895–915. 16. Ohmori, H. & Yoshii, M. (1977) J. Physiol. (London) 267, 429–463. 17. Byerly, L., Chase, P. B. & Stimers, J. R. (1985) J. Gen. Physiol. 85, 491–518. 18. Zhou, W. & Jones, S. W. (1995) J. Gen. Physiol. 105, 441–462. 19. Hess, P. & Tsien, R. W. (1984) Nature (London) 309, 453–456. 20. Kuo, C. C. & Bean B. P. (1993) Nature (London) 365, 258–262. 21. Sather, W. A., Yang, J. & Tsien, R. W. (1994) Curr. Opin. Neurobiol. 4, 313–323. 22. Prod’hom, B., Pietrobon, D. & Hess, P. (1989) J. Gen. Physiol. 94, 23–42. 23. Zamponi, G. W. & Snutch, T. P. (1996) Pflugers Arch. 431, 470–472. 24. Sheng, Z. H., Rettig, J., Cook, T. & Catterall, W. A. (1996) Nature (London) 379, 451–454. 25. Sheng, Z. H., Rettig, J., Takahashi, M. &Catterall, W.A. (1994) Neuron 13, 1303–1313. 26. Bezprozvanny, I., Scheller, R. H. & Tsien, R. W. (1995) Nature (London) 378, 623–626. 27. Bezprozvanny, I., Zhong, P., Scheller, R. H. & Tsien, R. W. (2000) Proc. Natl. Acad. Sci. USA. 97, 13943–13948. 28. Wiser, O., Bennett, M. K. & Atlas, D. (1996) EMBO J. 15, 4100–4110. 29. Wiser, O., Tobi, D., Trus, M. & Atlas, D. (1997) FEBS Lett. 404, 203–207. 30. Wiser, O., Trus, M., Hernandez, A., Renstrom, E., Barg, S., Rorsman, P. & Atlas, D. (1999) Proc. Natl. Acad. Sci. USA 96, 248–253.

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consistent with interfering of release-ready-vesicle assembly to an excitosome complex (30, 39). Moreover, inclusion of millimolar Ca2⫹-buffer EGTA did not affect the rate of secretion (39). These results are consistent with the idea that synaptic transmission is governed by the kinetics of divalent ions passing through the channel and not by Ca2⫹ diffusion and兾or cytoplasm free-Ca2⫹ buildup. Hence, the channel could act as the Ca2⫹ sensor protein and regulate the rapid secretion of channelassociated vesicles, operating upstream to a cytosolic Ca2⫹ sensor. Overall, these results help to explain evoked release in various external divalent ions. They propose a molecular rationalization for the efficient exocytosis of a distinct population of primed vesicles, highlighting the dynamic role of the channel in the stimulus-coupled event. Finally, a model is proposed in which rapid secretion of channel-associated vesicles perhaps may be initiated and terminated by conformational changes induced during a voltage step by divalent ions cruising the channel. The evidence suggests that the channel is the Ca2⫹ sensor protein of fast neurotransmitter release. This study was supported by the H. L. Lauterbach Fund (D.A.). 31. Charvin, N., L’eveque, C., Walker, D., Berton, F., Raymond, C., Kataoka, M., Shoji-Kasai, Y., Takahashi, M., De Waard, M. & Seagar, M. J. (1997) EMBO J. 16, 4591–4596. 32. Mochida, S., Sheng, Z. H., Baker, C., Kobayashi, H. & Catterall, W. A. (1996) Neuron 17, 781–788. 33. Rettig, J., Sheng, Z. H., Kim, D. K., Hodson, C. D., Snutch, T. P. & Catterall, W. A. (1996) Proc. Natl. Acad. Sci. USA 93, 7363–7368. 34. Tobi, D., Wiser, O., Trus, M. & Atlas, D. (1998) Recept. Channels 6, 89–98. 35. Yang, S. N., Larsson, O., Branstrom, R., Bertorello, A. M., Leibiger, B. & Leibiger, T. (1999) Proc. Natl. Acad. Sci. USA 96, 10164–10169. 36. Zhong, H., Yokoyama, C. T., Scheuer, T. & Catterall, W. A. (1999) Nat. Neurosci. 2, 939–941. 37. Trus, M., Wiser, O., Goodnough, M. C. & Atlas, D. (2001) Neuroscience 104, 599–607. 38. Atlas, D. (2001) J. Neurochem. 77, 961–974. 39. Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G. & Rorsman, P. (2001) Biophys. J. 81, 3308–3323. 40. Sheng, Z. H., Yokoyama, C. T. & Catterall, W. A. (1997) Proc. Natl. Acad. Sci. USA. 94, 5405–5410. 41. Schneider, T., Wei, X., Olcese, R., Costantin, J. L., Neely, A., Palade, P., Perez-Reyes, E., Qin, N., Zhou, J., Crawford, G. D., et al. (1994) Recept. Channels 2, 255–270. 42. Wu, L. G., Borst, J. G. & Sakmann, B. (1998) Proc. Natl. Acad. Sci. USA 95, 4720–4725. 43. Albillos, A., Neher, E. & Moser, T. (2000) J. Neurosci. 20, 8323–8330. 44. Saimi, Y. & Kung, C. (1982) Science 218, 153–156. 45. Heinemann, S. H., Terlau, H., Stuhmer, W., Imoto, K. & Numa, S. (1992) Nature (London) 356, 441–443. 46. Kim, M. S., Morii, T., Sun, L. X., Imoto, K. & Mori, Y. (1993) FEBS Lett. 318, 145–148. 47. Tang, S., Mikala, G., Bahinski, A., Yatani, A., Varadi, G. & Schwartz, A. (1993) J. Biol. Chem. 268, 13026–13029. 48. Yang, J., Ellinor, P. T., Sather, W. A., Zhang, J. F. & Tsien, R. W. (1993) Nature (London) 366, 158–161. 49. Bertram, R., Sherman, A. & Stanley, E. F. (1996) J. Neurophysiol. 75, 1919–1931. 50. Atlas, D., Wiser, O. & Trus, M. (2002) Cell Mol. Neurobiol. 21, in press.

PNAS 兩 March 19, 2002 兩 vol. 99 兩 no. 6 兩 3973

NEUROBIOLOGY

Unlike Syn1A, synaptotagmin either did not sense the conformational changes induced by the permeating ion or was unable by itself to modify the response of the channel to the divalents. Furthermore, because channel–Syn1A interaction could involve transmembrane domains, whereas the channel–synaptotagmin interaction is confined to the cytosolic domain(s), the latter is expected to be less susceptible to changes induced by the permeating ions.