Molecular determinants of Ca 2 /calmodulindependent regulation of Ca v 2.1 channels

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1 Molecular determinants of Ca 2 /calmodulindependent regulation of Ca v 2.1 channels Amy Lee*, Hong Zhou, Todd Scheuer*, and William A. Catterall* *Department of Pharmacology, University of Washington School of Medicine, Seattle, WA ; and Department of Pharmacology, Emory University School of Medicine, Atlanta, GA Contributed by William A. Catterall, October 28, 2003 Ca 2 -dependent facilitation and inactivation (CDF and CDI) of Ca v 2.1 channels modulate presynaptic P/Q-type Ca 2 currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca 2 involves calmodulin (CaM) binding to the 1 subunit ( 1 2.1). The molecular determinants for Ca 2 -dependent modulation of Ca v 2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of 1 2.1, the CaMbinding domain (CBD) and the IQ-like domain. In transfected tsa-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca 2 binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM 12 and CaM 34 were unable to bind CBD, whereas CaM 34 but not CaM 12 retained Ca 2 -dependent binding to the IQ-like domain. These findings support a model in which Ca 2 binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca 2 binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Ca v 2.1 in response to local Ca 2 increases (CDF) and negative regulation during more global Ca 2 increases (CDI). P /Q-type Ca 2 currents conducted by presynaptic Ca v 2.1 channels initiate neurotransmitter release at many central synapses. In nerve terminals, activity-dependent increases in intracellular Ca 2 ions cause an initial facilitation followed by a progressive inactivation of P/Q-type Ca 2 currents, which can alter synaptic efficacy (1 3). In transfected cells, Ca v 2.1 channels undergo a similar dual feedback regulation by Ca 2 that is mediated by calmodulin (CaM) (4, 5). CaM binds to the poreforming 1 subunit of Ca v 2.1 channels ( 1 2.1), causing an initial Ca 2 -dependent facilitation (CDF) and, on a longer time scale, Ca 2 -dependent inactivation (CDI) of these channels during repetitive stimuli (4, 5). How CaM binding to results in two opposing forms of channel regulation is not yet clear. A CaM-binding domain (CBD) in the cytoplasmic C-terminal tail of binds CaM in aca 2 -dependent manner, and deletion of this site diminishes but does not completely abolish CDF and CDI (4, 5). In addition, a second site on the N-terminal side of the CBD also participates in CaM regulation of Ca v 2.1 (6). This IQ-like domain is similar to the one that mediates CDI of L-type Ca 2 currents through Ca v 1.2 channels (7 9) but lacks the key Q residue at position 2 and hydrophobic residue at position 8 that characterize other IQ domains (10). Peptides corresponding to the IQ-like domain of bind CaM in vitro, and mutations in the IQ-like domain impair CDF of Ca v 2.1 channels (6, 7, 11). Moreover, fluorescence resonance energy transfer experiments show that CaM mutants incapable of binding Ca 2 interact with Ca 2 channels, suggesting that apocam is constitutively associated with Ca v 2.1 (12). In contrast, biochemical experiments that require stable association of CaM show strictly Ca 2 -dependent binding of CaM to the CBD and the IQ-like domain (4, 7). To clarify the role of the CBD and IQ-like domains in CDF and CDI, we analyzed the contributions of the CBD, the IQ-like domain, and the N- and C-terminal lobes of CaM in Ca 2 - dependent regulation of Ca v 2.1. Our results show that CDF and CDI result from a series of complex interactions between Ca 2 binding to specific lobes of CaM and distinct molecular contacts with the IQ-like domain and CBD. Experimental Procedures Molecular Biology constructs containing alterations of the CBD or IQ-like domain were generated by PCR from cdna encoding rat (rba) (13) lacking amino acids ( CBD ) has been described (5) IM-AA, IM-EE, and CBD/IM-AA were constructed by subcloning EcoRV/ PmlI fragments incorporating mutations at positions 1913 and 1914 into the corresponding sites of or CBD in a pbluescript SK shuttle vector. From this construct, a SgrAI/ MluI fragment containing the mutation(s) was subcloned into rba/pmt2xs t was constructed by amplifying the EcoRV/PmlI fragment with primers incorporating a stop codon after position 1965 and subcloning the truncated fragment into rba/pmt2xs. cdnas encoding CaM mutants (14), provided by John Adelman (Vollum Institute, Oregon Health and Science University, Portland, OR), were subcloned into BamHI sites of pcdna3.1 (Invitrogen) for mammalian cell expression or pgex4t1 for GST-fusion protein expression. His-tagged fusion constructs were generated by subcloning C-terminal fragments into BamHI sites of ptrchisa (Invitrogen). Electrophysiological Recording and Data Analysis. tsa-201 cells were grown to 70% confluence and transfected by the calcium phosphate method with an equimolar ratio of cdnas encoding WT or mutant 1 2.1, 2a, and 2 (15). For electrophysiological experiments, cells plated on 35-mm dishes were transfected with a total of 5 g of DNA including 0.3 g of a CD8 expression plasmid for detection of transfected cells. Twenty-four hours later, cells were plated at low density for electrophysiological recording. At least 48 h after transfection, cells were incubated with CD8-antibody-coated microspheres (Dynal, Oslo) to identify transfectants and washed in extracellular solution before recording. Whole-cell Ca 2 or Ba 2 currents were recorded with a List EPC-7 patch clamp amplifier driven by PULSE software (HEKA Electronics, Lambrecht/Pfalz, Germany). Leak and capacitive transients were subtracted by using a P/ 4 protocol. Extracellular recording solutions contained 150 mm Tris, 1 mm MgCl 2 and 10 mm CaCl 2 or BaCl 2. Intracellular solutions consisted of Abbreviations: CaM, calmodulin; CBD, CaM-binding domain; CDF, Ca 2 -dependent facilitation; CDI, Ca 2 -dependent inactivation. To whom correspondence should be addressed at: Department of Pharmacology, Box , University of Washington, Seattle, WA wcatt@u.washington.edu by The National Academy of Sciences of the USA NEUROSCIENCE cgi doi pnas PNAS December 23, 2003 vol. 100 no

2 120 mm N-methyl-D-glucamine, 60 mm Hepes, 1 mm MgCl 2,2 mm Mg-ATP, and 0.5 mm EGTA. The ph of all solutions was adjusted to 7.3 with methanesulfonic acid. Normalized tail current-voltage curves were fit with a single Boltzmann function: A/{1 exp[(v V 1/2 )/k] b}, where V is test pulse voltage, V 1/2 is the midpoint of the activation curve, k is a slope factor, A is the amplitude, and b is the baseline. Data analysis was done with IGOR PRO (WaveMetrics, Lake Oswego, OR). Binding Assays. GST-CaM and His fusion proteins were expressed in BL21 Escherichia coli and purified according to standard protocols. GST-CaM immobilized on glutathione agarose beads was incubated with 5 g of purified His fusion protein for 3hat4 C. After extensive washing, bound proteins were eluted and subjected to SDS/PAGE and immunoblotting with monoclonal anti-his or -GST antibodies. Results Roles of the CBD and the IQ-Like Domain in CDI. We constructed subunits with alterations in the IQ-like domain and the CBD (Fig. 1a) and analyzed CDI of Ca v 2.1 channels transfected in tsa-201 cells by using Ca 2 and Ba 2 as charge carriers and a relatively low concentration of EGTA (0.5 mm) in the recording pipette to minimize Ca 2 buffering (4, 5). Inactivation of WT and mutant channels was compared quantitatively as the ratio of the residual current amplitude at 800 ms to the peak current amplitude (I res /I pk ; Fig. 1b), and CDI was expressed as the difference between the average I res /I pk for Ca 2 and Ba 2. For 1 2.1, CDI is manifested as a faster decay of the Ca 2 current (I Ca ), and therefore, a significant decrease in I res /I pk for I Ca compared to I Ba ( , n 9 for I Ca vs , n 7 for I Ba ; CDI 0.27; P 0.01) subunits lacking the CBD ( CBD ) gave rise to channels with significantly reduced CDI (I res /I pk , n 7 for I Ca vs , n 9 for I Ba; CDI 0.10, P 0.01). We obtained similar results for subunits truncated immediately before the CBD ( t ; I res /I pk , n 10 for I Ca vs , n 9 for I Ba ; CDI 0.09, P 0.01). Despite the significant reduction in CDI for both CBD and t, I Ca still inactivated faster than I Ba, suggesting that a second site such as the IQ-like domain may also be involved in CDI. To assess the role of the IQ-like domain, alanine substitutions were made for the first two residues (IM) in this region ( IM- AA). These residues are critical for CaM regulation in conventional IQ domains in many other target proteins, including Ca v 1.2 channels (16, 17), and the IM-AA mutation in lowers the affinity for binding CaM by 30-fold in vitro (6). However, CDI was not significantly reduced in channels with IM-AA (I res /I pk , n 10 for I Ca vs for I Ba, n 6; CDI 0.21, P 0.11). Double mutant channels lacking the CBD and containing the IM-AA substitution ( CBDIM-AA ) exhibited significantly slower inactivation for I Ca and I Ba compared to WT (CDI 0.12, P 0.01; Fig. 1b), but the reduction in CDI was not greater than that caused by deletion of the CBD alone. Substitution of negatively charged glutamate residues for IM in the IQ-like domain had substantial effects on Ca v 2.1 function independent of Ca 2 ( IM-EE ; Fig. 1c). First, the IM-EE mutation caused a nearly 5-fold reduction in current, which necessitated the use of higher extracellular Ca 2 and Ba 2 concentrations to resolve effects on CDI. Second, IM-EE inactivated rapidly, with I Ca and I Ba decaying completely by 200 ms, a time point at which neither I Ca nor I Ba through WT is strongly inactivated. This marked enhancement of inactivation of IM-EE would partially occlude CDI because it exceeds the rate at which Ca 2 /CaM can induce CDI. However, comparison of the residual current amplitude at 50 ms revealed faster inactivation of I Ca than I Ba (Fig. 1c; I res /I pk , n Fig. 1. Contributions of the CBD and IQ-like domain to CDI. (a) Schematic showing constructs with alterations affecting the IQ-like domain (IM) and CBD in the C-terminal domain of 1 2.1; IM-AA containing AA in place of 1913 I 1914 M; t truncated after amino acid 1965; CBD lacking amino acids ; CBDIM-AA with both mutations; and IM-EE with EE in place of 1913 I 1914 M. (b) CDI of Ca v 2.1 channels containing 1 subunits with altered C termini. Normalized current traces are shown for currents evoked by 1-s test pulses from a holding voltage of 80 mv to 10 or 0 mv with 10 mm extracellular Ca 2 (black trace) or Ba 2 (gray trace), respectively. Intracellular solutions contained 0.5 mm EGTA. The current amplitude was measured at 800 ms (I res ) and normalized to the peak current amplitude (I pk ). Resulting I res /I pk values were averaged (n 5 10) and plotted ( SEM). Asterisks indicate significant differences between CDI [(I res /I pk for I Ca ) (I res /I pk for I Ba )] for mutant compared to WT channels (P 0.05). (c) Enhanced inactivation of IM-EE. Normalized currents evoked by 0.5-s test pulses from 80 to 10 (I Ca, black trace) or 0 mv (I Ba, gray trace). I res was measured at 50 ms, and I res /I pk was plotted as in b. 5 for I Ca vs , n 5 for I Ba ; P 0.06), suggesting that CDI remains at least partially intact after the IM-EE mutation. Therefore, our results support a prominent role for the CBD, but not the IQ-like domain, in CDI of Ca v 2.1. Roles of the CBD and the IQ-Like Domain in CDF. To analyze CDF of Ca v 2.1, we used two protocols similar to those used to assess facilitation of synaptic transmission (Fig. 2). During trains of repetitive stimuli, I Ca increases to a new steady level, whereas I Ba remains relatively constant (Fig. 2a). During paired-pulse experiments, I Ca evoked after a conditioning prepulse (P2) is greater than I Ca elicited before the prepulse (P1) (Fig. 2f ). These two measures of CDF provide complementary information: the paired-pulse protocol shows CDF evoked by a single episode of sustained Ca 2 influx, and the cumulative increase of I Ca during trains of stimuli also reflects the stability cgi doi pnas Lee et al.

3 Fig. 2. Contributions of the IQ-like domain and CBD to CDF. (a d) Facilitation of I Ca during 100-Hz trains of pulses from 80 to 10 or 0 mv, as indicated in the voltage protocol shown above the panels. Test current amplitudes ( SEM, n 4) were normalized to the first in the train, and every third point is plotted against time. (e) Averaged normalized current amplitudes between 0.5 and 1s,(I n/i 1). Asterisks indicate significant difference between mutant and WT channels (P 0.05). (f i) CDF in paired-pulse protocols, as indicated above the panels, with extracellular 10 mm Ca 2 and intracellular 0.5 mm EGTA. Tail current amplitudes were measured on repolarization to 40 mv, normalized to the mean tail current after P1 pulses between 50 to 70 mv, and plotted against test pulse voltage. Insets show representative P1 and P2 currents. Plotted points are mean SEM for P1 ( ) and P2 ( ) for (n 5), IM-AA (n 5), CBD (n 4), and CBDIM-AA (n 7). (j) Means for I Ca or I Ba of P2 max /P1 max, where PX max is the mean value of tails after pulses between 50 and 70 mv. of CDF in the interval between pulses. Our previous results show that the stability of CDF is strongly enhanced by Ca 2 and CaM (5). Compared to WT Ca v 2.1 (Fig. 2f), deletion of the CBD reduced the paired-pulse facilitation of I Ca (Fig. 2 h and j). Similarly, deletion of the CBD caused a partial loss of facilitation measured in the repetitive pulse protocol (Fig. 2 c and e). By contrast, there was no significant facilitation of IM-AA or CBDIM-AA in paired-pulse experiments (Fig. 2 g, i, and j) and no difference between facilitation of I Ca and I Ba in repetitive pulse experiments (Fig. 2 b, d, and e). These results demonstrate a primary requirement for the IQ-like domain in CDF and a modulatory role for the CBD in this process. Fig. 3. Requirement for N- and C-terminal EF-hands of CaM for CDI. (Upper) Schematic of CaM. (Lower) CDI for Ca v 2.1 channels cotransfected with CaM mutants. Normalized current traces are shown for 1-s test pulses from a holding voltage of 80 mv to 10 or 0 mv for I Ca (black traces) or I Ba (gray traces) with 0.5 mm EGTA intracellular. Mean I res /I pk values ( SEM; n 5 12) for I Ca or I Ba as in Fig. 1b for the indicated CaM constructs. Asterisks indicate significant differences from WT CaM (P 0.01). Distinct Functions of the N- and C-Terminal EF-Hands of CaM in CDI. We have shown previously that CDI but not CDF is blocked by 10 mm EGTA, a high-affinity but slow Ca 2 chelator, whereas both processes are blocked by 10 mm BAPTA, a rapid chelator (5). The distinct Ca 2 sensitivities for the two processes indicate that CDF is triggered by local Ca 2, which is intercepted rapidly by BAPTA, whereas CDI is induced by global Ca 2 elevations, which are removed more slowly by EGTA. The molecular basis for this difference between CDF and CDI could reside in the N- and C-terminal lobes of CaM, which bind Ca 2 ions with different affinities (18, 19). To test this, we used CaM constructs containing alanine substitutions for critical aspartate residues that impair Ca 2 coordination in the paired EF-hands of the N-terminal lobe (CaM 12 ), the C-terminal lobe (CaM 34 ), or both lobes of CaM (CaM 1234 ) (Fig. 3) (14). Although coexpression of Ca v 2.1 with CaM or CaM 34 did not influence CDI during 1-s depolarizing pulses, CaM 12 virtually eliminated the difference in inactivation between I Ca and I Ba (Fig. 3). As expected, there was also substantially reduced CDI when CaM 1234 was expressed, although its effect was less complete than CaM 12 (Fig. 3). These results point to a specific requirement for the N-terminal EFhands of CaM for CDI. Dual Requirement for the N- and C-Terminal Lobes of CaM in CDF. In a previous study (6), CaM 12 supported CDF in paired-pulse experiments, whereas CaM 34 did not, leading to the proposal that CDF was mediated primarily by the C-terminal EF-hands of CaM. We further investigated the function of the N- and C-terminal lobes of CaM by using the paired-pulse and repetitive-stimulation protocols (Fig. 4). As in the previous work, we found nearly complete loss of CDF in the paired-pulse protocol (Fig. 4 h and j) and in the repetitive-stimulation protocol (Fig. 4 c and e) with the CaM 34 mutant. However, we also found a significant impact of the CaM 12 mutant on facilitation in both protocols. In the paired-pulse protocol, facilitation of I Ca was substantially reduced by CaM 12, but facilitation of I Ba was reduced to a similar extent (Fig. 4 g and j; see also ref. 6). In the repetitive pulse protocol, there was nearly complete loss of facilitation (Fig. 4 b and e), and differences between I Ca and I Ba with CaM 12 were not significant ( , n 13 vs NEUROSCIENCE Lee et al. PNAS December 23, 2003 vol. 100 no

4 Fig. 4. Requirement for N- and C-terminal EF-hands for CDF. (a d) CDF during 100-Hz trains of depolarizations as in Fig. 2 a d [mean SEM for CaM (n 11), CaM 12 (n 13), CaM 34 (n 9)], and CaM 1234 (n 7). (e) Averaged normalized current amplitudes between 0.5 and 1sasinFig. 2e. (f i) CDF during paired-pulse protocols measured as in Fig. 2 e i [mean SEM for CaM (n 10), CaM 12 (n 6), CaM 34 (n 7), and CaM 1234 (n 5)]. (j) Mean values for P2 max /P1 max for I Ca or I Ba determined as in Fig. 2j. Asterisks indicate significant differences from WT (P 0.05). 0.04, n 13; P 0.22). CaM 1234 also caused nearly compete loss of CDF in both protocols (Fig. 4 d, e, i, and j). Together, these results confirm an absolute requirement for Ca 2 binding to the C-terminal lobe of CaM for CDF and reveal a significant role for the N-terminal lobe as well. Differential Binding of the IQ-Like Domain and CBD to CaM. To determine whether differences in CaM-binding to the CBD and IQ-like domain contribute to their different functional effects, we measured the direct interaction of the CBD and IQ-like domains with CaM, CaM 12, and CaM 34 in a binding assay that detects stable, high-affinity CaM-binding interactions. Using GST-tagged CaMs immobilized on glutathione agarose beads, we observed Ca 2 -dependent binding of His-tagged CBD and IQ-like domain peptides, as reported (4, 7, 11) (Fig. 5 a and b). Neither CaM 12 nor CaM 34 associated with the CBD, indicating that functional EF-hands in both the N- and C-terminal lobes are required for stable Ca 2 -dependent CaM interaction with this site (Fig. 5c). By contrast, CaM binding to the IQ-like domain was prevented by inactivation of the N- but not C-terminal EF-hands (Fig. 5c), consistent with results of yeast-two-hybrid Fig. 5. Binding of CaM to the IQ-like domain and the CBD. (a) His-tagged C-terminal fragments including the IQ-like domain (IM, amino acids ), CBD ( ), or an upstream sequence ( ) used as a negative control. (b)ca 2 -dependent binding of His-tagged C-terminal fragments (His) to GST-tagged CaM (input, GST) immobilized on glutathione agarose in the presence of 2 M Ca 2 or 10 mm EGTA was detected by immunoblot with anti-his antibodies. (c) Differential binding of CaM 12 and CaM 34 to the IQ-like domain and CBD. GST-tagged CaM 12 or CaM 34 was used to pull-down His-tagged C-terminal fragments in 2 M Ca 2 as in b. (d) Model for CDI and CDF based on sequential interactions of the IQ-like domain and CBD with CaM. analyses (20). These results eliminate the possibility that blockade of CDF by CaM 34 (Fig. 4) was caused by its inability to interact with the IQ-like domain. Evidently, Ca 2 binding to the N-terminal lobe is sufficient for stable CaM binding to the IQ-like domain but not for induction of CDF. Discussion Specific Roles of the CBD and IQ-Like Domain in Ca 2 -Dependent Regulation of Ca v 2.1. Our previous results showed that Ca v 2.1 channels undergo prominent CDF and CDI and identified the CBD as a CaM-binding motif that contributes to these forms of cgi doi pnas Lee et al.

5 regulation (4, 5). The experiments presented here also demonstrate a significant loss of CDI in Ca v 2.1 channels lacking the CBD (Fig. 1) and provide the first evidence for an important role of the CBD in CDF measured in paired-pulse and repetitive stimulation paradigms (Fig. 2). Deletion of the CBD selectively in CBD or by truncation of the C-terminal domain in t caused a similar reduction in CDF and CDI. Evidently, the CBD has functionally significant interactions with CaM in both CDF and CDI, but these are not the exclusive interactions that mediate these regulatory processes because measurable CDF and CDI remain in CBD-deletion mutants. Our results also extend the work of DeMaria and colleagues, who discovered a major role for the IQ-like domain in CDF of Ca v 2.1 (6). Like these authors, we find that mutation of the first two amino acid residues in the IQ-like domain to alanine (IM-AA) completely prevents CDF (Fig. 2 b and g), indicating that the IQ-like domain is necessary for CDF of Ca v 2.1 channels. In contrast to these results with CDF, we did not resolve a significant effect of the IM-AA mutation on CDI, in agreement with previous work (6). Because the IM-AA mutation causes a 30-fold reduction in affinity for CaM (6), these results suggest that CDF requires high-affinity binding of CaM that is prevented by the IM-AA mutation. In contrast, CDI either does not require the IQ-like domain or requires only low-affinity interactions that are retained in the IM-AA mutant. Mutation of the same two amino acid residues in the IQ-like domain to glutamate (IM-EE) completely blocks CaM binding to the IQ-like domain and dramatically accelerates voltagedependent inactivation of I Ba (Fig. 1c and ref. 6). This result establishes an important role for these amino acid residues in control of voltage-dependent inactivation in the absence of Ca 2. Acceleration of voltage-dependent inactivation to the extent observed for the IM-EE mutation would also cause the channel to inactivate before Ca 2 /CaM could initiate CDI, regardless of the role of the IQ-like domain in this process. We have shown that similar rapid, voltage-dependent inactivation of Ca v 2.1 caused by coexpression of auxiliary 1b rather than 2a subunits largely occludes CDI, even though these subunits have no direct role in CDI itself (5). Despite this potential difficulty, we were able to measure residual CDI of the IM-EE mutant at an early (50 ms) time point (Fig. 1c). These results and the lack of effect of the IM-AA mutation on CDI shown here and in previous work (6) indicate that the IQ-like domain does not have a prominent role in CDI of Ca v 2.1 channels. Although it is possible that the IQ-like domain does have a significant role in CDI, its impact is not revealed by the IM-EE or IM-AA mutations that have been analyzed to date. Our results differ from those of DeMaria et al. (6), who found that deletions of the CBD did not impact either CDF or CDI. These different results may in part be due to methodological differences. Our CDF protocols measure an increase in peak I Ca rather than an increased rate of activation of I Ca as in previous work (6), which may require more stable binding of CaM through interaction with the CBD. Our CDI experiments show that deletion of the CBD significantly reduced but did not abolish CDI (Fig. 1 and ref. 4), indicating that other regions of the channel contribute to CaM-dependent conformational changes leading to CDI. The functional roles of the CBD in CDI and CDF that we have detected by using the rat brain (rba isoform) may be less apparent with the human isoform used in the previous work (6). Although the C-terminal regions, including the IQ-like domain and the CBD, are quite similar in the human and rat sequences, functionally significant differences may exist within these conserved sequences or in other parts of the channel that could influence channel modulation by CaM (21, 22). Distinct Roles of the N- and C-Terminal Lobes of CaM. CaM mutants can disrupt Ca 2 -dependent modulation of Ca 2 channels by displacing endogenous CaM binding to the channel and preventing Ca 2 -dependent conformational changes leading to CDF and CDI (6 8). However, interpretation of these results is complicated by the presence of endogenous CaM at sufficient concentration to give both CDF and CDI. In this situation, the lack of effect of a mutant CaM may reflect inability to bind Ca v 2.1, thereby allowing endogenous CaM to function normally. Alternatively, a CaM mutant may have no apparent effect because, although it does interact with Ca v 2.1, it causes CDF and CDI that are indistinguishable from endogenous CaM. Moreover, some CaM mutants may have effects that are independent of their inability to bind Ca 2. Mutation of the N-terminal EF-hands of CaM completely blocks CDI (Fig. 3 and ref. 6). These results indicate that CaM 12 does interact with Ca v 2.1 and prevent the action of endogenous CaM but cannot cause CDI itself. Because we do not detect stable high-affinity binding of CaM 12 in our biochemical experiments (Fig. 5c), reversible, low-affinity interactions of overexpressed CaM 12 are apparently sufficient to disrupt CDI by endogenous CaM. By contrast, CaM 34 has no effect on CDI (Fig. 3 and ref. 6), but our binding studies show that it can bind to the IQ-like domain (Fig. 5). These results indicate that CaM 34 is fully active in CDI and therefore that Ca 2 binding to the N-terminal EF-hands of CaM is sufficient for CDI. Mutation of the C-terminal lobe of CaM completely prevents CDF in both paired-pulse and repetitive-stimulation protocols (Fig. 4), as in previous work using different stimulus paradigms (6). CaM 34 can bind to the IQ-like domain in the presence of Ca 2 (Fig. 5c), so the block of CDF by CaM 34 evidently results from its inability to bind Ca 2 to EF-hands 3 and 4 and induce conformational changes within the IQ-like domain that favor CDF. We also found that mutation of the N-terminal lobe of CaM significantly reduces facilitation measured in the pairedpulse protocol and nearly completely prevents facilitation during trains of stimuli (Fig. 4). In contrast to the loss of CDF with CaM 34, CaM 12 reduces facilitation in the presence of Ca 2 but also decreases the limited facilitation observed when Ba 2 is the charge carrier. Ba 2 may bind weakly to the N-terminal EFhands of CaM and partially support facilitation. In fact, Ba 2 - dependent inactivation of Ca v 1.2 has been observed and proposed to be due to Ba 2 binding to CaM (23, 24). In any case, our results indicate that Ca 2 binding to EF-hands 1 and 2 is required for maximal facilitation when measured as an increase in peak I Ca in paired-pulse or repetitive-stimulation protocols, perhaps because Ca 2 -binding to EF-hands 1 and 2 increases the overall affinity of CaM binding to the CBD. A Revised Molecular Model for CDF and CDI of Ca v 2.1. Lobe-specific regulation is a general feature of functional interactions of CaM with many targets (14, 20, 25, 26). Although a number of ion channels are modulated by direct interactions with CaM (27), Ca v 2.1 channels are unusual in that CaM binding mediates two opposing forms of modulation that are kinetically and mechanistically distinct (5). Our results support and extend a model (6) in which CDF and CDI result from Ca 2 binding to specific lobes of CaM and sequential changes in the interaction of Ca 2 /CaM with the IQ-like domain and CBD. Although our biochemical experiments do not detect stable CaM binding to the IQ-like domain or the CBD in the absence of Ca 2 (Fig. 5b), the millisecond kinetics of CDF imply that CaM must be reversibly bound at resting Ca 2 levels in the cell. In our revised model (Fig. 5d), voltage-gated Ca 2 influx through Ca v 2.1 would promote local Ca 2 binding to the C-terminal lobe of preassociated CaM, which initiates or strengthens interaction with the IQ-like domain. More global increase in Ca 2 leads to binding of Ca 2 ions to the N-terminal lobe of CaM and also induces a conforma- NEUROSCIENCE Lee et al. PNAS December 23, 2003 vol. 100 no

6 tional change that allows binding of fully Ca 2 -liganded CaM to the CBD. Binding of Ca 2 /CaM to the CBD then enhances CDF and initiates CDI. According to this model, when preassociated CaM is replaced by CaM 34,Ca 2 influx initiates Ca 2 binding to the N-terminal lobe of CaM, which supports only CDI. When CaM is replaced by CaM 12,Ca 2 influx initiates Ca 2 binding only to the C-terminal lobe of CaM that permits partial induction of CDF. Considerable progress has been made toward understanding where CaM interacts with Ca v 2.1 and other voltage-gated Ca 2 channels and how this interaction can modulate channel function (4 6, 11, 17, 28 30). However, it is becoming increasingly clear that Ca 2 -dependent regulation of Ca v 2.1 is complex, involving Ca 2 -dependent and -independent interactions of CaM with multiple sites on the subunit. It is possible that CaM regulation of Ca v 2.1 involves dynamic interactions between the CBD, IQ-like domain, and potentially other sites, as has been proposed for the multiple CaM-binding sequences in the ryanodine receptor (31, 32). Moreover, emerging evidence shows that Ca 2 sensors in addition to CaM are likely to participate in Ca v 2.1 regulation in neurons (33, 34). How such interactions are ultimately transduced into physiological forms of channel modulation is an intriguing question to resolve in future structural and functional analyses. We thank Elizabeth Sharp and Hong Sun for assistance in constructing mutants, Dr. John Adelman for his generous gift of CaM mutants, and Dr. Bertil Hille (Department of Physiology and Biophysics, University of Washington) for comments on a draft of the manuscript. This work was supported by National Institutes of Health Grants NS22625 (to W.A.C.) and NS (to A.L.) and National Research Service Award NS10645 (to A.L.). 1. Forsythe, I. 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