Calcium channel inactivation: Possible role in signal transduction and Ca2+ signaling

Cell Calcium 38 (2005) 223–231

Calcium channel inactivation: Possible role in signal transduction and Ca2+ signaling Martin Morad a,b,∗ , Nikolai Soldatov a,b a

Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road NW, Washington, DC 20057, USA b National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 22124, USA Received 20 June 2005; accepted 28 June 2005 Available online 11 August 2005

Abstract Voltage gated Ca2+ channels are major routes for the entry of intracellular Ca2+ coupled to membrane depolarization that appear to vary greatly with respect to their voltage dependence and kinetics. Such variability maybe in part related to the attached signaling properties of the channel, in addition to the transport of calcium. In the present review we consider the possible role of calcium-dependent inactivation of Cav 1.2 in Ca2+ signal transduction and signaling of calcium release from the cardiac sarcoplasmic reticulum. We explore the specific roles of Ca2+ -sensing calmodulin-binding domains of the C-terminal tail (LA and K) of the channel in mediating Ca2+ -induced Ca2+ release and signal transduction. Our experiments point to an intriguing possibility that the C-terminal tail of Cav 1.2 may translocate the Ca2+ signal as a part of inactivation mechanism and the corresponding voltage-gated rearrangement of the C-terminus. We show how a dynamic and transient regulation, in a Ca2+ -dependent manner, defines molecular events including Ca2+ release and signaling of cAMP-responsive element-binding protein (CREB)-dependent transcription. We propose that such Ca2+ -dependent C-tail translocation that also initiates the channel inactivation, may have evolved specifically for the Cav 1.2 channel. © 2005 Elsevier Ltd. All rights reserved. Keywords: Ca channel inactivation; Cav 1.2 channel; Ca signaling; Signal transduction; C-terminal of Ca channel

Voltage-gated Ca2+ channels generate transient increase in intracellular Ca2+ concentration ([Ca2+ ]i ) activated by membrane depolarization. Cellular responses associated with transient rise of cytosolic Ca2+ range from sarcomeric contraction, to cell growth and proliferation to synaptic transmission. At least 10 different types of Ca2+ channels, subdivided into three major families, Cav 1 (L type), Cav 2 (N, P/Q and R type), and Cav 3 (T type) have been thus far described [1]. In this review we shall focus on one of the first and most evolved member of Ca2+ channel family, the L-type Cav 1.2. This channel is widely expressed in the human body and couples membrane depolarization to many essential cellular processes including transcription activation [2,3], muscle contraction [4,5], synaptic plasticity [6] and exocytosis [7]. The pore function of the Cav 1.2 channel is encoded in the ∗

Corresponding author. Tel.: +1 202 687 8453; fax: +1 202 687 8458. E-mail address: [email protected] (M. Morad).

0143-4160/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2005.06.027

Cav 1.2␣1 subunit (known also as ␣1C ) that is also the target of Ca2+ channel blockers [8]. Heterologous expression experiments show that for the channel to become fully functional, Cav 1.2 requires association of the ␣1C subunit with the two other major accessory subunits: the cytoplasmic ␤ and the extracellular ␣2 which is in turn bound via disulphide bond to the transmembrane peptide ␦. Co-expression of these subunits generates a fully functional ␣1C /␤/␣2 ␦ channel that exhibits most if not all the properties of the native Cav 1.2 channel. Some properties of the native channel are also affected by splice variants of ␣1C [9,10]) and ␤ subunits [11,12]. Cytoplasmic domains of Cav 1.2 channel have also evolved fairly intricate calmodulin (CaM) dependent signaling mechanism. It is now generally believed that all L-channel types include a mechanism that provides for the negative feedback inhibition of the Ca2+ current (Ca2+ induced inactivation), modulated in part by the cytoplasmic domains of the Ca2+

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Fig. 1. (A) Effect of the replacement of Ba2+ for Ca2+ as the charge carrier on kinetics of the currents through the conventional ␣1C,77 /␤1a /␣2 ␦ channel. Shown are representative current traces evoked by depolarization to +10 mV (Ba2+ ) or +20 mV (Ca2+ ) applied from Vh = −90 mV. (B) Effect of removal (␣1C,86 ) or inhibition (␣1C,IS-IV ) of voltage-dependent slow inactivation on the mode of the Ba2+ current decay. Shown are traces of the Ba2+ current through the ␣1C,86 /␤1a /␣2 ␦ and ␣1C,IS-IV /␤1a /␣2 ␦ channels evoked by +10 mV depolarization applied from Vh = −90 mV. Striking similarity between the Ca2+ current through the ␣1C,77 channel and Ba2+ current through the ␣1C,78 channel is one of the evidences that CDI targets slow inactivation mechanism. All current traces were scaled to the same maximum amplitude.

channel. Such a mechanism of Ca2+ -dependent inactivation, resulting in acceleration of inactivation of the Ca2+ current in response to the rise of intracellular Ca2+ [13], was first identified in cardiac L-type channels [14,15], and is thought to have a critical role in cardiac EC coupling [16]. Similar experiments on the recombinant channel also show that replacement of extracellular Ca2+ by Ba2+ eliminates Ca2+ dependent inactivation (Fig. 1A). Nevertheless, two-exponentials are required to adequately fit the Ba2+ current carried through the “conventional” ␣1C,77 /␤1a /␣2 ␦ channel. Quantitative analysis suggests that both fast (τ f = 105.7 ± 8.4 ms) and slow (τ s = 590 ± 48 ms) components of the Ba2+ current are comparably large (If = 44 ± 2.8% and Is = 37.3 ± 2.8%, respectively; n = 38, Xenopus oocytes expression system). In addition, there was also a significant sustained non-inactivating component of the Ba2+ current (18.6 ± 1.1% of the total current). The same analysis applied to the Ca2+ transporting channel showed that both the slow (τ s = 254 ± 15 ms; If = 3.6 ± 0.5%) and sustained components (Io = 4.3 ± 0.6) were virtually eliminated, leaving the prominent fraction of ICa (If = 93.5 ± 0.5%, n = 28) with a single-component and accelerated decay kinetics (τ s = 24.6 ± 1.2 ms). Thus, replacement of Ba2+ by Ca2+ accelerates the inactivation rate of the fast component of the current and essentially eliminates the slow component. Taken together, these results suggest that Cav 1.2 inactivates by two different mechanisms, the slower of which is especially sensitive to permeating Ca2+ . Mutation analysis of Cav 1.2 allowed to identify molecular determinants of Ca2+ and voltage dependent inactivation. 1. Determinants of Ca2+ dependent inactivation Fig. 1B shows superimposed traces of Ba2+ currents through two different mutants of the ␣1C,77 channel that either lack the slow component of inactivation (␣1C,86 channel)

or where the channel fails to inactivate by slow mechanism (␣1C,IS-IV channel). The ␣1C,86 channel [17] has the 80-amino acid locus in the second quarter of the carboxyl-terminal tail of ␣1C,77 replaced with essentially different 81 amino acids encoded by the alternatively spliced combination of exons 40–43 coding for this region of the Cav 1.2␣1 subunit gene [18]. We called this locus the “Ca2+ -sensing domain” (CSD). Nevertheless, transplanting the entire C-terminal tail of ␣1C into the ␣1G subunit failed to convey Ca2+ dependent inactivation to T-type calcium channel [19], suggesting that CSD is sufficient but not essential for Ca2+ dependent inactivation. Since our earlier studies had suggested that Ca2+ channel inactivation could be impaired by single mutation of A752T in the human fibroblast ␣1C Ca2+ channel transcript [20], and similarly, Val1504 in IVS6 of the rabbit cardiac ␣1C were shown to be critical for the channel inactivation [21], a new mutant the ␣1C,IS-IV channel [22] was generated by simultaneous mutation of four highly conserved amino acids (S405I in IS6, A752T in IIS6, V1165T in IIIS6 and I1475T in IVS6) of transmembrane segments S6, that are believed to form the cytoplasmic end of the pore of Cav 1.2. Because these mutations are located in positions −1 (IS6) and −2 (IIS6–IVS6), the corresponding structure in the ␣1C,77 channel was named the “annular determinant of slow inactivation” (ADSI) reflecting the specific arrangement of crucial amino acids with regards to the ion-conducting pore. Critical for present narration, it is important to note that both ␣1C,86 and ␣1C,IS–IV channels, though completely deprived of Ca2+ dependent inactivation, have fairly different inactivation kinetics. The very fact that two distantly located determinants, ADSI in the pore region and CSD in the proximal locus of the C-tail, are independently crucial for Ca2+ dependent inactivation indicates that not only their specific chemical structure, but also their mutual interaction is essential. Several observations support this suggestion: First, large changes in voltage-dependence of activation and inactivation of the Ba2+

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current were observed when comparing ␣1C,86 and ␣1C,77 channel [17] suggesting that voltage sensors responsible for channel gating and located in the plasma membrane could sense the changes caused by amino acid replacements in CSD of the cytoplasmic tail. Second, when comparing the single channel properties of ␣1C,77 , with those of ␣1C,86 and its derivatives, significant decrease in the unitary Ba2+ current of the mutant were observed [23], which points to an interaction between the CSD and the pore region. Taken together, these data imply that slow voltage-dependent inactivation and Ca2+ dependent inactivation irrespective of charge carrier (Ba2+ versus Ca2+ ) require interaction of CSD with the pore region, where ADSI is located. This interaction may dynamically react to membrane voltage supporting state-dependent transitions of the channel between resting, open and inactivated conformations.

2. Role of CaM in Ca2+ dependent inactivation It is now generally accepted that Ca2+ dependent inactivation is associated with two adjacent motifs in the C-terminal. These two loci, originally named L (1572–1598) and K (1595–1651), were identified in the C-tail of Cav 1.2 and were shown to be independently important for Ca2+ dependent inactivation [24,25]. Subsequent to the strategy developed by Adelman’s group for the Ca2+ -dependent K+ channel [26] it became possible to narrow down the K locus and identify the IQEYFRKFKKRK (1624–1635, a part of the K locus) mutant as a CaM-binding site crucial for Ca2+ dependent inactivation [27]. This idea was further confirmed by the same authors (Z¨uhlke et al., 1999) and two other groups [28,29]. These experiments, although clearly demonstrated that CaM is preassociated with Cav 1.2 at the IQ containing K domain, did not fully clarify the mechanism of Ca2+ dependent inactivation [30,31]. Critical in evaluating the role of the carboxyl tail of ␣1C in Ca2+ dependent inactivation is the finding that the LA motif (1571–1599, RIKTEGNLEQANEELRAIIKKIWKRTSMK) of the locus L binds CaM in the resting state, when [Ca2+ ]i is very low (50–100 nM) as well as when [Ca2+ ]i exceed 1–5 ␮M levels. IQ motif, on the other hand, appears to bind the Ca2+ -CaM complex during the Ca2+ conducting state of the channel [32,33,34]. Recent NMR studies of interactions between Ca2+ -CaM and peptides corresponding to amino acids 1538–1692 (somewhat similar to L + K loci) and 1596–1692 (K locus) show conformational changes in Ca2+ -CaM upon binding to K locus independent of the presence of L peptide [35], suggesting possibly a functional switch between CaM associated with L and K that is regulated by the transient rise of [Ca2+ ]i . The Ca2+ dependent inactivation mechanism may also be effectively eliminated by the expression of the dominantnegative mutant CaM1234 that has lost all of its high affinity Ca2+ binding motifs, but retains binding affinity to the CaM-binding sites [27,36]. Fig. 2 illustrates this point by

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showing Ba2+ and Ca2+ currents through the ␣1C,77 /␤1a /␣2 ␦ channel expressed in Xenopus oocytes coexpressed with CaMWT (A) or CaM1234 (B). It can be clearly seen that Ca2+ current exhibits greater accelerated decay kinetics and 1/3 lower conductance than IBa , in the presence of CaMWT (Fig. 2A). In contrast, when the dominant negative CaM1234 is co-expressed with the channel, kinetics of inactivation was not significantly accelerated, even though the maximum amplitude of the current was greatly reduced (Fig. 2B). It is somewhat puzzling that if CaM was so critical in Ca2+ dependent inactivation, then why have CaM inhibitors been so ineffective in altering the kinetics of Cav 1.2? One likely possibility is that Ca2+ dependent inactivation may take place at a site closely associated with the internal permeating site of the channel. Indeed, experiment with photorelease of “caged” [Ca2+ ]i (50 ␮M within 0.2 ms) in dorsal root ganglion neurons though accelerating the kinetics of the Ca2+ conducting channel, failed to have a similar effect on the Na+ current through the channel [37]. Further, we found that angiotensin-triggered Ca2+ release failed to accelerate the kinetics of inactivation of Ba2+ transporting ␣1C,77 when the channel was coexpressed with the AT1A receptor in Xenopus oocytes (Fig. 3). These experiments further showed that 1 ␮M angiotensin transiently stimulated Ca2+ release via the G-protein/IP3-dependent pathway (Fig. 3A and B) [38], consistent with the idea that CaM is pre-associated with Cav 1.2 [30,34]).

3. Apocalmodulin and “CAM-glue” hypothesis Since apo-CaM has the ability to cross-link polypeptide chains [39,40]), we considered the possibility that apo-CaM might integrate the Ca2+ sensitive domain of the C-terminal into a cytoplasmic polypeptide bundle underlying the pore by transiently cross-linking the ␣1C subunit C-tail to a docking site near the pore region thus stabilizing the resting conformation of the channel [41–43]). In this scheme as the channel opens, the permeating Ca2+ binds to CaM, thus inhibiting the apo-CaM mediated cross-linking and releasing the CSD from the disintegrated bundle. Over-expression of CaM1234 does not compromise the molecular glue function of CaM, but eliminates the sensitivity of the complex to Ca2+ ions (Fig. 2B). For this reason the Ca2+ current through the ␣1C /CaM1234 channel inactivates with both fast and slow mechanisms similar to those of the Ba2+ -conducting channel (Fig. 1A). According to this “CaM-glue” hypothesis, Ca2+ dependent inactivation is a manifestation of the Ca2+ -induced release of the CSD-CaM complex from the resting confirmation of the cytoplasmic polypeptide bundle that protects it from the cytoplasmic Ca2+ and keeps it accessible only to the pore-permeating Ca2+ . It is tempting to suggest that the CSD-CaM bundling in the vicinity of ADSI defines the slow inactivation. Obviously, such interaction is absent in the T-channels that inactivate

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Fig. 2. Effect of Ca2+ insensitive mutant CaM1234 on Ba2+ and Ca2+ currents through the Cav 1.2 channel. Shown are 250 ms traces of the Ba2+ and Ca2+ currents evoked by +10 mV depolarization applied from Vh = −90 mV to Xenopus oocytes that express ␣1C,77 /␤1a /␣2 ␦ channels in the presence of the wild-type CaM (A) or CaM1234 (B).

rapidly with either Ca2+ or Ba2+ as the charge carrier. If our hypothesis is correct, then uncoupling of the CSD-CaM complex from the bundling should eliminate CDI. This experiment has been carried out using transient anchoring of the C-terminal tail of the ␣1C,77 subunit to the plasma membrane by fusion of the plextrin homology (PH) domain protein to the last amino acid of the tail [3,43]. Fig. 4 shows that the anchoring of the ␣1C,77 subunit C-terminal tail did distinctly prolong plateau at approximately a half-maximum of the Ba2+ (A) and Ca2+ current (B). Thus, uncoupling of the C-tail by the plasma membrane anchoring caused complete inhibition of CDI and slow voltage-dependent inactivation of the channel similar to the mutation of the ADSI (Fig. 1B). An interesting distinction from the electrophysiological phenotype of the ␣1C,IS-IV channel, however, was a shift of the I–V relationship to more positive potentials. Because these changes occur without structural alterations of the ADSI or the CSD, and are completely reversible upon the release of the PH-anchored ␣1C,77 C-tail, stimulated by the hydrolysis of the PH-domain phosphatidylinositol bisphosphate (PIP2 ) upon activation of phospholipase C (Fig. 4A, lower trace), it is reasonable to assume that the plasmamembrane anchoring of the tail interferes with the functional folding of the cytoplasmic polypeptide bundle vis-a-vis the pore region (including ADSI) that eliminates Ca2+ dependent inactivation.

Fig. 4. Effect of the anchoring of the ␣1C subunit C-terminal tail in the plasma membrane on inactivation of the Ba2+ (A) and Ca2+ current (B). The PH domain was genetically fused to the last amino acid of the ␣1C,77 C-tail. The ␣1C,77 -(PH)C /␤1a /␣2 ␦ channel was expressed in COS1 cells together with EGF receptor. Currents were activated by +20-mV depolarization applied from Vh = −90 mV. To release PH-domain, serum-deprived (24 h) cells were exposed to EGF (100 ng/ml).

Fig. 3. Lack of acceleration of the Ba2+ current by intracellular Ca2+ release. The Cav 1.2 channel coupled with AT1A angiotensin receptor was expressed in Xenopus oocytes. (A) Time-dependence and reversibility of the angiotensin effect on IBa through the ␣1C,77 /␤1a /␣2 ␦ channels expressed alone (filled squares) or co-expressed with AT1A receptors (open squares). Amplitudes of IBa were measured in response to 250-ms test pulses to +20 mV applied with 30-s intervals and normalized to the maximum IBa in the absence of angiotensin. Arrows indicate the time of application of bath solutions containing shown concentration of angiotensin (in ␮M). (B) Traces of IBa through the ␣1C,77 /␤1a /␣2 ␦ channel co-expressed with AT1A receptor recorded before (lower trace) and 1.5, 2 and 3 min after application of 1 ␮M angiotensin to stimulate intracellular Ca2+ release [38].

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Fig. 5. Evidence of the voltage-gated reversible rearrangements of the ␣1C subunit C-terminal tail. (A) Phase-contrast image of expressing COS1 cells with the shadow of patch pipette (scale bar, 8 ␮m), and schematic diagrams depicting the arrangement of the ECFP and EYFP FRET partners in the ␣1C subunit of the expressed (EYFP)N -␣1C,77 -(ECFP)C /␤1a /␣2 ␦ channel. Yellow box refines the region of the plasma membrane where FRET was measured. Ba2+ current was evoked by 600-ms depolarizations to +20 mV from Vh = −90 mV. Applied voltage protocol is presented above the current trace. (B) and (C) Ratio of corrected FRET images sequentially recorded with FRET cube at resting (−90 mV) and inactivated states of the channel (+40 mV) and associated with a reversible voltage-gated rearrangement between the fluorophores. The time-windows for the −90 and +40-mV FRET acquisitions are marked by red and green horizontal bars above the Ba2+ current trace. Scaling bar applies to both images. Note that in every case the Ba2+ current recordings provided evidence of the channel state achieved prior to FRET image acquisitions (marked by bars). FRET at Vh = −90 mV was recorded for the same duration of time before and after the depolarization pulse.

4. Voltage-gated conformational rearrangement of the ␣1C C-terminal tail and its role in Ca2+ signal transduction and Ca2+ release from the RyRs Fluorescence resonance energy transfer (FRET) in voltage clamped cells provides optical measurements under statedependent conditions showing that the shorter N-terminal tail of the ␣1C subunit (in the presence of ␤) does not rearrange vis-a-vis the plasma membrane in response to voltage gating [43]. In sharp contrast the ␣1C C-terminal tail shows voltage-dependent conformational rearrangements (Fig. 5). Measurements of corrected FRET between the enhanced yellow (EYFP) and cyan fluorescent proteins (ECFP), genetically attached, respectively to the N- and C-termini of ␣1C,77 showed no significant effect on steady-state current or its kinetics, but there was substantial increase in FRET signal accompanying the inactivated state of the channel (Fig. 5B) that was fully reversible upon transition of the channel into the resting state (Fig. 5C) [3]. 4.1. Signal Transduction Since C-tail anchoring eliminated Ca2+ dependent inactivation, we investigated whether the voltage-gated conformational rearrangements of the ␣1C C-tail plays a role in Ca2+ signal transduction that, e.g. is utilized in Ca2+ induced activation of cAMP-responsive element-binding protein (CREB)-dependent transcription. We took advantage

of the test system based on the measurement of interaction between KID and KIX domains of CREB and coactivator CREB-binding protein (CBP) under voltage-clamp conditions by monitoring FRET between (EYFP)-KID and (ECFP)-KIX, both containing nuclear localization sequences [44]. In perforated whole clamped cells, where the integrity of the cytoplasmic content of the cell is intact (Fig. 6A) no activation of CREB transcription was observed when the ␣1C C-tail was uncoupled by anchoring it to the plasma membrane (Fig. 6B, panel a) in spite of large sustained inward Ca2+ current and the corresponding increase in [Ca2+ ]i detected by Fluo4 fluorescence (panel b). Release of the ␣1C C-tail by activation of PIP2 hydrolysis upon activation of M1AchR (Fig. 6C) at –90 mV caused significant elevation of [Ca2+ ]i that was not utilized by the cell for CREB transcription activation (Fig. 6C, panel a) until a depolarizing pulse to +20 mV was applied and the C-tail of the L-channel permitted to move (Fig. 6D). This experiment provided compelling evidence that large inward Ca2+ current or the subsequent rise in intracellular Ca2+ does not lead to CREB transcription activation, unless it is accompanied by the conformational rearrangement of the ␣1C subunit C-terminal tail that provides the precise targeting of the Ca2+ signal transduction (Fig. 6D, panel a) [3]. Recent experiments indicated that the both co-expression of CaM1234 , or mutation of the LA site in the ␣1C subunit C-tail inhibit CREB transcription activation in the absence of tail anchoring. Thus, the native bundling of the ␣1C C-tail

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Fig. 6. Evidence that Ca2+ signal transduction by the Cav 1.2 channel to activate CREB transcription is mediated solely by the voltage-gated mobility of the ␣1C subunit C-tail and is not directly associated with the Ca2+ current. CREB activation was examined under perforated patch conditions. (A) Top panel, Phase-contrast image of the COS1 cell with a shadow of patch pipette. The cell was expressing the ␣1C,77 -(PH)C /␤1a /␣2 ␦ channel with the membrane-trapped ␣1C subunit C-tail, type 1 muscarinic Ach receptor (to release the C-tail in response to activation by Ach), and EYFP-labeled KID domain of CREB and ECFPlabeled KIX domain of CREB-binding protein. Lower panel, representative trace of the Ca2+ current (20 mM Ca2+ in bath medium) evoked by depolarization to +20 mV from Vh = −90 mV showing the sustained component of Ca2+ conductance due to the C-tail anchoring. (B) Plasma membrane anchoring of the ␣1C subunit C-tail (see schematic diagram) inhibits CREB activation in spite of large Ca2+ current evoked by +20 mV depolarization. (a) A 100-ms images of FRET between EYFP-KID and ECFP-KIX were recorded at the end of the 12th +20 mV depolarization step applied every 10 s for 1 s from −90 mV. (b) an increase of [Ca2+ ]i detected by Ca2+ indicator Fluo4. (C) ACh-stimulation of the M1AChR receptor caused activation of IP3 -dependent Ca2+ release but did not show substantial activation of CREB transcription at Vh = −90 mV. (D) CREB-dependent transcription activation was initiated only when a +20-mV depolarization was applied to Cav 1.2 with the C-terminal tail release from the plasma membrane to assume functional conformation.

near the pore region, mediated by CaM and the LA locus, in the resting state of the channel (−90 mV) are crucial for Ca2+ signal transduction. 4.2. Signaling of cardiac ryanodine receptors by C-terminal tail There is general agreement that Ca2+ release from cardiac sarcoplasmic reticulum is almost exclusively triggered by influx of Ca2+ through the Cav 1.2 channel. The most compelling observation in this respect was the finding that suppression of Ca2+ current by removal of Ca2+ or influx of Na+ and Ba2+ through the channel shortly (∼50 ms) before activation of Ca2+ channels completely suppressed Ca2+ release [45]. In addition, Ca2+ release could be terminated rapidly on premature termination of Ca2+ influx either by deactivation of ICa or by step depolarization of membrane to ECa [5]. Since influx of Ca2+ on the exchanger was 100 times less effective in triggering Ca2+ release than ICa [46], it was concluded that Ca2+ channel had privileged access to RyRs. This concept was strongly supported by the finding that dialysis of high concentration of Ca2+ buffers (EGTA and Fura 2) failed to significantly alter the cross signaling between Ca2+ channels and RyRs [47,48]. Occurrence of spontaneous or triggered Ca2+ sparks [49] in confined spatial domains on activation of myocytes and their resistance to high concentrations of Ca2+ buffers and their development into Ca2+ stripes at sarcomeric spacing associated with t-tubular system [50] further solidified the concept that Ca2+ signaling occurs in the microdomains associated with the DHPR/RyRs complex

[51,52]. The Ca2+ induced Ca2+ release (CICR) occurring in molecular domains of DHPR/RyRs is essentially a Ca2+ amplifying system. The most unexpected aspect of CICR gain is its voltage dependence shortly at −30 mV, where influx of Ca2+ through the channel is very small [53,54]. This voltage dependence of amplification factor (gain) of CICR is not readily expected from a strictly Ca2+ dependent Ca2+ release mechanism, but is more consistent with additional step that may require voltage dependent protein-protein interaction or conformational change. To test for such a possibility, peptide fragments of Ca2+ sensing domains of the C-terminal tail of ␣1C77 were introduced into the cardiac myocytes. To prevent possible interaction of these peptides with the native Ca2+ channel C-terminal tail/RyR complex, we chose to image development of spontaneous Ca2+ sparks and their activation at different voltages and subsequent local rises in [Ca2+ ]i in atrial myocytes, using a 2D rapid confocal imaging system. We chose rat atrial myocytes because these cells are mostly free of t-tubular system, effectively preventing the central RyRs from close association with DHPRS. Dialysis of atrial myocyte with LA peptide containing the apocalmodulin site produced a three- to four-fold increase in spontaneous spark frequency in the central region where RyRs are mostly free of DHPRs, “naked”. In sharp contrast, K peptide containing the CaM-binding IQ motif failed to have a significant effect on the frequency of spontaneous sparks, suggesting that LA peptide sensitizes the RyRs and increases their opening probability [55]. This was consistent with the finding that rate of caffeine induced Ca2+ release was increased and the delay in its activation strongly abbreviated

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in LA, but not K peptide dialyzed myocytes [55]. Segment of replacement of apocalmodulin domain of LA peptide by another set of amino acids (LM peptide) totally eliminated the LA peptide-induced enhancement of spark frequency, suggesting direct interaction of this site with RyRs. Quantification of gain of CICR also suggested that only LA, but not K peptide increased the gain (Ca/ICa ) of central release sites at −30 mV, but not at +20 mV [55]. This finding is consistent with the idea that when Ca2+ influx through the L-type Ca2+ channel is minimally activated, LA domain of the C-terminal tail may directly interact with RyRs sensitizing it to CICR. One possibility to consider is that the LA peptide may bind CAM associated with RyRs, relieving CAM inhibitory effect on the RyRs. Irrespective of the exact molecular mechanism involved, it is quite clear that specific molecular domains on the C-terminal tail of the ␣1C subunit cross communicate with RyRs in regulating Ca2+ signaling. 4.3. Hypothesis linking Ca2+ channel activation and inactivation to signal transduction We propose that activation of the Ca2+ current saturates

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Cav 1.2. Thus, an attempt to determine functional stoichiometry of CaM and Cav 1.2 using CaM fused to the ␣1C C-tail [62] may be somewhat misleading because this approach is based on assumption that an individual Cav 1.2 molecule is selfsufficient in regulation. It is highly likely that CaM attached to C-tails of neighboring channel molecules in Cav 1.2 clusters may compete for the same docking site. It is intriguing to speculate that such clustering may be critical in Ca2+ cross signaling between DHPRs and RyRs in t-tubular system of cardiac myocytes where global cellular RyR/DHPR ratios is generally assumed to be ∼5/1. In conclusion, accumulated body of data is sufficient to suggest that influx of Ca2+ ions is not directly involved in Ca2+ signal transduction. It is the ␣1C subunit C-termial tail voltage gated conformational rearrangements that is essential to deliver Ca2+ ions to downstream signaling targets in a transiently caged, CaM-bound form. This process is intimately associated with Ca2+ induced inactivation of the channel that releases the Ca2+ /CaM loaded C-tail from the polypeptide bundle underlying the cytoplasmic end of the pore. In the cardiac muscle, this process may dynamically support molecular signalling within microdomains of the RyRs.

Ca2+ -binding sites of CaM, disrupting the cross-linking function of apo-CaM, which in turn disrupts the resting-state folding of CSD in the pore region. The CSD is then released from the pore bundle moving the Ca2+ -CaM complex associated with IQ domain of the tail to the downstream target, where some Ca2+ is released to higher affinity targets. Loss of Ca2+ then increases the affinity of CaM to the LA site, which can then promote the return of the tail to its resting position requiring energy (consistent with the long-lasting recovery of ICa from inactivation by strong hyperpolarization). This process properly reinstalls CSD in the bundle in preparation for a new cycle of Ca2+ -CaM shuttling to begin. This model explains many aspects of the channel regulation, including apparent acceleration of inactivation of the Cav 1.2 channel in response to strong intracellular Ca2+ release in cardiac myocytes. However, this effect does not contradict the notion that CSD is not available to the cytoplasmic Ca2+ . It is the inhibition of the return of the tail to the resting conformation that may be responsible for this effect. Indeed, dissociation of the Ca2+ -CaM complex must be suppressed by strong intracellular Ca2+ release, which will “freeze” the C-tail in an unfolded conformation. The other possibility is that the cross-linking of the C-tail by CaM to alternative targets would compete with the return of the tail to the channel’s docking site. Such alternative targeting may explain the tendency of the Cav 1.2 channel to form clusters that was shown in various cells including sympathetic neurons [56], hippocampal pyramidal neurons [57] and cardiac myocytes [58,59,60]. The average size of a cluster of human recombinant Cav 1.2 expressed in HEK 293 cells was found to be ≈40 [61]. Interestingly, genetic mutations in the L and/or K regions that inhibit Ca2+ dependent inactivation and CaM binding [23] effectively eliminate cluster formation of

Acknowledgements Supported: NIH grant # Ro1 16152 (to M.M.) and NIA Intramural Research Program (to NMS).

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