The IQ Motif is Crucial for Cav1.1 Function

Ca2+-dependent modulation via calmodulin, with consensus CaM-binding IQ motif playing a key role, has been documented for most high-voltage-activated Ca2+ channels. The skeletal muscle Cav1.1 also exhibits Ca2+-/CaM-dependent modulation. Here, whole-cell Ca2+ current, Ca2+ transient, and maximal, immobilization-resistant charge movement (Qmax) recordings were obtained from cultured mouse myotubes, to test a role of IQ motif in function of Cav1.1. The effect of introducing mutation (IQ to AA) of IQ motif into Cav1.1 was examined. In dysgenic myotubes expressing YFP-Cav1.1AA, neither Ca2+ currents nor evoked Ca2+ transients were detectable. The loss of Ca2+ current and excitation-contraction coupling did not appear to be a consequence of defective trafficking to the sarcolemma. The Qmax in dysgenic myotubes expressing YFP-Cav1.1AA was similar to that of normal myotubes. These findings suggest that the IQ motif of the Cav1.1 may be an unrecognized site of structural and functional coupling between DHPR and RyR.


Introduction
Calcium entering the cell through voltage-gated Ca 2+ channels plays an important role in mediating a wide variety of cellular events and includes feedback processes that regulate activity of the channel itself. The Ca 2+ -dependent modulation of channel activity mediated by the Ca 2+ -binding protein calmodulin (CaM) is found in many ion channels including the Ca v 1 family [1]. Ca 2+ -dependent inactivation (CDI) of Ca v 1.2 is mediated by CaM, and its structural determinants have been assigned to the proximal region of the C-terminus of Ca v 1.2 [1,2]. Three domains have been identified within this region: a Ca 2+ binding EF-hand motif, a CaM-tethering site, and a CaM-binding IQ motif. The EFhand motif, located ∼16 residues beyond the end of the last transmembrane segment (IVS6), is absolutely necessary for CDI. The CaM-tethering site, which consists of both preIQ 3 and IQ motifs, resides 50 amino acids downstream from the EF-hand motif and binds Ca 2+ -free CaM (apo-CaM) at resting [Ca 2+ ] i . The IQ motif resides downstream from the EFhand motif and the pre-IQ 3 domain, and it binds Ca 2+ -CaM. When the interaction of CaM with either of these domains is compromised, CDI is reduced or eliminated [1,2].
Recently, it has been demonstrated that the skeletal muscle L-type Ca 2+ channel (Ca v 1.1) also displays CDI mediated by CaM and that CaM associates with Ca v 1.1 in vivo [3]. The initial 200 amino acids of the C-terminus of the Ca v 1.1 are highly conserved and contain the abovedescribed domains including the IQ motif. CaM binding to the IQ motif of Ca v 1.2 channel has been shown to be necessary for CDI, and the mutation of the isoleucine (I1624) and glutamine (Q1625) to alanines (AA) in the IQ motif of Ca v 1.2 resulted in ablation of CDI and significant reduction of apoCaM binding to Ca v 1.2 [1,2,4]. Whether the IQ motif in Ca v 1.1 plays a similar role remains to be determined.
In the present work, myotubes cultured from normal and dysgenic (lacking endogenous Ca v 1.1) mice were used to investigate the role of the IQ motif in the function of Ca v 1.1. The results presented demonstrate that the IQ motif in the C-terminus of Ca v 1.1 is critical for function of Ca v 1.1 as a voltage sensor as well as Ca 2+ channel. Furthermore, the results indicate that the IQ motif may be a previously unrecognized site of protein-protein interaction between Ca v 1.1 and the skeletal muscle ryanodine receptor (RyR1) and may play a role in skeletal muscle excitation-contraction (EC) coupling.

Molecular Biology.
The coding sequence of yellow fluorescent protein-(YFP-) tagged Ca v 1.1 channel (YFP-Ca v 1.1) was a gift from Dr. K. Beam and is described in detail elsewhere [5]. The residues isoleucine (I) and glutamine (Q) at codons 1529-1530 of rabbit Ca v 1.1 [6] were substituted with alanine (A) using the QuikChange II mutagenesis kit (Stratagene, La Jolla, CA), using the YFP-Ca v 1.1 as a template. The construct YFP-Ca v 1.1 AA was verified by restriction digest analysis and sequencing.

Cell
Cultures. Primary myotubes were cultured from normal or dysgenic newborn mouse skeletal muscle as previously described [3]. For confocal microscopy purposes, primary cultures of myotubes were plated onto 35 mm culture dishes with integral no. 0 glass coverslip bottoms (MatTek) instead of Primaria dishes. Approximately one week after plating, dysgenic myotubes were injected with expression plasmids (cDNAs) encoding either YFP-Ca v 1.1 or YFP-Ca v 1.1 AA at concentrations of 0.2 μg/μL, respectively. In experiments assessing the effects of CaM on Ca 2+ transients, normal myotubes (∼one week in culture) were injected with expression plasmids encoding CaM wt or CaM 1234 (gift of Dr. Yue) and green fluorescent protein (pEGFP-C1, BD Biosciences Clontech, CA) at concentrations of 0.1 and 0.02 μg/μL, respectively. Successfully transfected myotubes were identified 36-48 hours after injection by their yellow or green fluorescence under UV illumination.

Electrophysiology.
Patch pipettes were constructed of borosilicate glass and had resistances of 1.8-2.5 MΩ when filled with the standard internal solution, which contained (in mM) 145 Cs-aspartate, 10 Cs 2 -EGTA, 5 MgCl 2 , and 10 HEPES (pH 7.4 with CsOH). The external solution contained (in mM) 145 tetraethylammonium chloride (TEA-Cl), 10 CaCl 2 , 0.003 tetrodotoxin, and 10 HEPES (pH 7.4 with TEA-OH). The holding potential was -80 mV, and test pulses were preceded by a 1-s prepulse to -30 mV to inactivate endogenous T-type Ca 2+ currents. Recorded membrane currents were corrected off line for linear components of leakage and capacitance by digitally scaling and subtracting the average of 10 preceding control currents, elicited by hyperpolarizing voltage steps (30 mV amplitude) from -50 mV. Ca 2+ currents were normalized by linear cell capacitance (expressed in pA/pF). Values for G max , the maximal Ca 2+ conductance, were obtained by fitting the measured currents according to the following equation: where I peak is the peak current activated at the test potential V , V R is the extrapolated reversal potential, V 1/2 is the potential for half-maximal activation of the Ca 2+ conductance, and k is a slope factor. The fraction of current remaining at the end of an 800 ms test pulse (r 800 ) was determined by dividing the current remaining at the end of test pulse by the peak current, and this ratio was used to quantify the level of inactivation For measurements of charge movement, 0.5 mM Cd 2+ and 0.1 mM La 3+ were added to the external solution to block Ca 2+ currents. Charge movements were elicited in response to a prepulse protocol that consisted of a 1-s prepulse to −30 mV and a subsequent 40 ms repolarization to a pedestal potential (−50 mV), followed by a 25 ms depolarization to +40 mV. The maximum amount of charge that can be moved (Q max ) was obtained by integrating the charge movement current at test potential of +40 mV. Linear leak and capacity currents were subtracted on line using −P/4 delivered from the holding potential (−80 mV) before each pulse. Charge movements were normalized to total cell capacitance (nC/μF).
To measure relative changes in voltage-gated Ca 2+ release from the SR, the Ca 2+ indicator K 5 -Fluo-3 (0.5 mM) (Molecular Probes) was included in the pipette solution. After rupture of the cell membrane and entry into the whole cell configuration, cells were allowed to dialyze for about 5 min before recording in order to achieve adequate loading with indicator dye. Fluorescent emission was measured by a photomultiplier system (Biomedical Instrumentation Group, University of Pennsylvania). The set of filters used to record the fluorescent signal from Fluo-3 was as follows: excitation band-pass filter of 470/20 nm; dichroic long-pass mirror (510 nm); emission long-pass filter of 520 nm. After rupture and dye loading into the cell, the baseline fluorescence (F base ) was monitored. The increase in fluorescent signal during depolarization was expressed as ΔF/F, where ΔF represents the increase in fluorescence above baseline fluorescence (ΔF = F transient − F base ), and F is F base . Peak fluorescence during each test pulse was plotted as a function of test potential V and fitted according to the following equation: where (ΔF/F) max is the maximal fluorescent change, V F1/2 is the potential for half-maximal activation of the Ca 2+ transient, and k F is a slope factor. All recordings were performed at room temperature (∼20 • C), and data are reported as mean ± SEM; n indicates the number of myotubes tested. Data sets were statistically compared by an unpaired, two-sample Student's t-test, with a confidence interval of at least 95%. fluorophore. Emissions of YFP were recorded in single-track configuration with a long-pass filter of 530 nm (Chroma, Rockingham, Vermont). Fluorescence signals were analyzed by the 510 LSM Image Examiner software (Zeiss, Thornwood, New York).

2.5.
Immunocytochemistry. Primary cell cultures were plated onto 35 mm culture dishes with integral no. 0 glass coverslip bottoms (MatTek). Myotubes expressing constructs of Ca v 1.1 were identified by fluorescence. The cultures will be fixed with 100% methanol at −20 • C for a minimum of 20 min. Cells were then incubated for 1 hour in PBS (phosphatebuffered saline) containing 1% BSA (bovine serum albumin) and 10% goat serum to block unspecific labeling. After 3 washes with PBS/BSA (.2%), cell cultures were incubated with specific primary antibody against the RyR1 (34C, Developmental Studies Hybridoma Bank (DSHB), UI) (dilution 1 : 4000) overnight at 4 • C. Cells were washed out 3 times with PBS/BSA (.2%), followed by 1 hour of incubation with secondary antibody conjugated with Alexa 568 (at final dilution 1 : 5,000, goat anti-rabbit IgG, Invitrogen). Cells were then washed 3 times with PBS/BSA (.2%) to remove unbind secondary antibody and assessed with a confocal microscope.

Results
Ca 2+ -binding ability of CaM does not affect skeletal muscle EC coupling. First, I addressed the question whether the Ca 2+binding ability of CaM plays any role in skeletal muscle EC coupling. Overexpressed mutant CaM which does not bind Ca 2+ (CaM 1234 ) can displace approximately 70% of endogenous CaM, as reflected by abolishment of CDI of Ca v 1.1 [3]. However, overexpression of either CaM wt or CaM 1234 in normal myotubes did not significantly affect either current-voltage (I/V) relationship (Figure 1(a)) or voltage-gated Ca 2+ release from SR as indicated by similar peak fluorescence-voltage relationship (ΔF/F-V ) in comparison with uninjected normal myotubes (Figure 1(b)). This result suggests that either the Ca 2+ -binding ability of CaM or CaM itself does not play a role in skeletal muscle EC coupling. However, CaM associates with Ca v 1.1 in vivo [3] and that indicates the possibility that CaM may still serve as a structural subunit of Ca v 1.1, that is, that interaction between CaM and Ca v 1.1 can stabilize the DHPR complex. By doing so, it may also ensure proper structural and functional coupling between DHPR and RyR1.
Therefore, I examined whether CaM association with Ca v 1.1 is necessary for its function as a voltage sensor for EC coupling. The IQ motif of Ca v 1.1 has been shown to bind CaM similar to IQ motifs of Ca v 1.3 and Ca v 2 channels [7]. Introduction of the mutation IQ/AA in the IQ motif of the cardiac L-type Ca 2+ channel (Ca v 1.2) resulted in abolishment of CDI and significant reduction of apoCaM binding to Ca v 1.2 [2,4]. Thus, corresponding IQ motif mutation in the C-terminus of Ca v 1.1 was obvious place to start.
The mutation (IQ/AA) in the CaM-binding site of Ca v 1.1 disables function of Ca v 1.1 as a Ca 2+ channel and voltage sensor for EC coupling. I introduced the IQ/AA mutation in the C-terminus of Ca v 1.1 and investigated how this  Figure 2(a) shows Ca 2+ currents mediated by YFP-Ca v 1.1 expressed in dysgenic myotube. The fraction of current remaining at the end of the pulse (r 800 ) displayed a Ushaped voltage dependence (data not shown), consistent with a current-dependent inactivation process. In such a process, the extent of inactivation varies in proportion with the amplitude of the inward calcium current, which in turn depends on the number of conducting channels and the electrochemical driving force on calcium. Inactivation was minimal at a test potential of +10 mV, as reflected by an r 800 value of 0.9 ± 0.08 (n = 7), and maximal at a test potential of +40 mV, as reflected by a minimum r 800 value of 0.74 ± 0.03 (n = 7). Correspondingly, the Ca 2+ current attained its maximum conductance at +40 mV (Figure 1(c)). Thus, Ca 2+ currents mediated by YFP-Ca v 1.1 displayed a current-dependent inactivation process, currentvoltage (I-V ) relationship (Figure 2(c)), and maximal Ca 2+ ion conductance (G max = 166 ± 18 nS/nF; n = 7) similar to the endogenous Ca v 1.1 of normal myotubes [3]. These results suggest that YFP fused to the N-terminus of Ca v 1.1 does not interfere with channel function.
In contrast, dysgenic myotubes expressing YFP-Ca v 1.1 AA (Figures 2(b) and 2(c)) displayed either very small (<1pA/ pF) or no measurable Ca 2+ currents. This is a very dramatic and surprising result considering that the corresponding mutation (IQ/AA) in the IQ motif of Ca v 1.2 resulted only in ablation of CDI but did not affect the I-V relationship of Ca 2+ currents mediated by Ca v 1.2 [2]. Further voltagegated Ca 2+ currents and SR Ca 2+ release were measured simultaneously from dysgenic myotubes expressing YFP-Ca v 1.1 AA and compared with recordings from uninjected normal myotubes and normal myotubes overexpressing CaM wt or CaM 1234 . The voltage-gated Ca 2+ release from SR was completely abolished in dysgenic myotubes expressing YFP-Ca v 1.1 AA (Figure 2(d)).
The loss of Ca v 1.1 AA function could be a result of several scenarios such as that mutation caused misfolding of protein and insufficient membrane targeting or that protein-protein interaction between RyR1 and Ca v 1.1 was significantly disturbed. If the latter possibility is the case, this result suggests that either the IQ motif itself or association of CaM with Ca v 1.1 is necessary for orthograde signaling from Ca v 1.1 to RyR1, which underlies skeletal muscle EC coupling.
The IQ/AA mutation does not prevent proper targeting of Ca v 1.1 into sarcolemma. The severe loss of function, abolished Ca 2+ current and orthograde signaling mediated by the Ca v 1.1 AA , could be explained by compromised targeting of Ca v 1.1 to the T-SR junction as a result of incomplete protein folding. Figure 3 shows confocal images of yellow fluorescence from a dysgenic myotube expressing either YFP-Ca v 1.1 or YFP-Ca v 1.1 AA . Expression of YFP-Ca v 1.1 (a) or YFP-Ca v 1.1 AA (b) resulted in the appearance of small yellow fluorescence puncta located near the cell surface. The small puncta correspond to groups of Ca v 1.1 localized to T-SR junctions; these puncta are similar in size and distribution to those of Ca v 1.1 foci revealed by immunohistochemistry [11]. There is a similar staining of the membrane and distribution of puncta in both myotubes, suggesting that both constructs are likely targeted to T-SR junctions.
To confirm targeting of YFP-Ca v 1.1 AA to the sarcolemma, the Q max was measured at +40 mV ( Figure 4). The Q max in dysgenic myotubes expressing Ca v 1.1 AA (5.9±0.5 nC/μF; n = 27) was similar to that of normal myotubes (5.5 ± 0.4 nC/μF; n = 16), but significantly larger (P < 0.001) than in dysgenic myotubes alone (2.5 ± 0.2 nC/μF; n = 18). This finding suggests that IQ/AA mutation in Ca v 1.1 did not prevent the protein from being properly targeted or undergoing voltagedependent conformational changes, which strongly suggest proper folding as intramembrane segment S4 of Ca v 1.1 is responsible for voltage-dependent movement.
To further confirm Ca v 1.1 AA proper targeting into T-SR junctions and site of EC coupling, I investigated colocalization of Ca v 1.1 and RyR1. Dysgenic myotubes expressing either YFP-Ca v 1.1 or YFP-Ca v 1.1 AA (yellow fluorescence: YFP was artificially assigned as green) were incubated with specific primary antibody against the RyR1 followed by incubation with secondary antibody conjugated with Alexa 568 (red fluorescence). Colocalization of green and red fluorescence results in yellow pattern suggests colocalization of YFP-Ca v 1.1 and RyR1 in T-SR junctions in vivo (see Together these results suggest that the IQ/AA mutation is not likely to affect protein folding within membrane. Furthermore, much more drastic alternation or deletions in Ca v 1.1 sequence did not have such dramatic effects [12,13]. Taking altogether, the loss of both ionic Ca 2+ current and skeletal muscle EC coupling in Ca v 1.1 AA along with charge movement similar to normal myotubes suggests that the IQ motif of the Ca v 1.1 may be unrecognized site of proteinprotein interaction between Ca v 1.1 and RyR1 and play a role in both orthograde and retrograde signaling.

Discussion
The present study provides new information about the skeletal muscle L-type Ca 2+ channel (Ca v 1.1). Specifically, the data demonstrate in vivo that the IQ motif in the Cterminus of Ca v 1.1 is critical for function of Ca v 1.1 as a voltage sensor as well as a Ca 2+ channel. Furthermore, the results indicate that the IQ motif, in addition to II-III loop, may be a previously unrecognized site of protein-protein interaction between Ca v 1.1 and RyR1 and, thus, may play a role in skeletal muscle EC coupling.
Ca v 1.1 is localized in regions of the T-tubular membrane that are closely apposed to the sarcoplasmic reticulum (i.e., the T-SR junction), and the primary role of Ca v 1.1 is to serve as the voltage sensor for skeletal muscle EC coupling. The second protein that plays a major role in this process is the skeletal muscle ryanodine receptor (RyR1). RyR1 is localized in junctional SR membrane and functions as calcium release channel. The mechanism of signal transmission between Ca v 1.1 and RyR1 is still incompletely understood, but the most accepted view is that they are mechanically coupled and interact with each other through protein-protein interaction (orthograde and retrograde signaling). Orthograde signaling is the signal from Ca v 1.1 to RyR1, in which movement of the voltage sensors in Ca v 1.1 trigger opening of RyR1 and release of Ca 2+ from the SR (EC coupling). Retrograde signaling is communication from RyR1 to Ca v 1.1, in which RyR1 somehow increases the amount of L-type Ca 2+ current mediated by Ca v 1.1 [8,9].
The Ca 2+ conductance of Ca v 1.1 channel is not necessary for functional excitation-contraction coupling between RyR1 and Ca v 1.1; however, a direct protein-protein interaction between these two proteins in multiple sites is. It has been shown that cytoplasmic loops of Ca v 1.1 and several regions of RyR1 play important role for normal physiological EC coupling in skeletal muscle [10,[14][15][16][17]. It has been also shown that protein-protein interaction between RyR1 and Ca v 1.1 is necessary for Ca v 1.1 display of Ca 2+ conductance (retrograde signaling) [8]. It is clear that there are multiple contact sites between RyR1 and Ca v 1.1 and not all of them are recognized and understood, yet. The most investigated region of contact between RyR1 and Ca v 1.1 in Ca v 1.1 is II-III cytoplasmic loop, but other regions play a role [14,15].
In the present experiments, normal myotubes and dysgenic myotubes expressing either YFP-Ca v 1.1 or YFP-Ca v 1.1 AA were used to examine the role of the IQ motif in both functions of Ca v 1.1, as a voltage sensor in EC coupling and Ca 2+ channel. The primary cultures of skeletal muscle myotubes provide a natural cellular environment for Ca v 1.1. First, I examined whether a fusion of YFP to Ca v 1.1 would interfere with its function. The Ca 2+ currents mediated by YFP-Ca v 1.1 displayed an I-V relationship similar to the endogenous Ca v 1.1 [3], suggesting that YFP fused on the Nterminus of Ca v 1.1 does not interfere with its channel function, as was also shown by others [5]. Endogenous Ca v 1.1 also exhibits CaM-mediated Ca 2+ -dependent inactivation (CDI) [3]. The Ca 2+ currents mediated by YFP-Ca v 1.1 also displayed current-dependent inactivation similar to the CDI of endogenous Ca v 1.1, further supporting observation that fusion of YFP with Ca v 1.1 does not interfere with channel function.
Second, I examined how IQ/AA mutation in Ca v 1.1 will affect its function. Surprisingly, the intriguing finding of the present study was that dysgenic myotubes expressing YFP-Ca v 1.1 AA displayed either very small or no measurable Ca 2+ currents. Significant decrease or abolishment of Ca 2+ current through Ca v 1.1 could have resulted from improper targeting or folding of the protein. If Ca v 1.1 AA was retained inside of myotubes due to incorrect folding and targeting, neither Ca 2+ currents nor Q max would be obtained. The absence of Ca 2+ currents in some of the dysgenic myotubes expressing YFP-Ca v 1.1 AA would suggest both. However, even though the Ca 2+ current was absent, Q max comparable with normal myotubes was observed. The Q max measured in dysgenic myotubes expressing Ca v 1.1 AA was significantly larger (P < 0.001) than in dysgenic myotubes alone, but similar to that of normal myotubes measured here and to the Q max measured in dysgenic myotubes expressing various constructs of wt Ca v 1.1 at the similar experimental conditions elsewhere [6,7]. The amount of Q max in dysgenic myotubes expressing YFP-Ca v 1.1 AA suggests that IQ/AA mutation in Ca v 1.1 did not prevent the protein from being properly targeted and that protein can undergo voltage-dependent conformational changes. The size of small measurable Ca 2+ currents measured in some (6 out of 14) of the dysgenic myotubes expressing Ca v 1.1 AA (<1pA/pF) was similar to L-type Ca 2+ currents measured in dyspedic (lacking a functional gene of RyR1) myotubes [7], suggesting a loss of retrograde signaling from RyR1. Endogenous Ca v 1.1 channels are present in sarcolemma of the dyspedic myotubes in similar density as in normal myotubes, as was demonstrated by comparable Q max (dyspedic: 4.0 ± 1.4 nC/μF; normal: 6.4 ± 2.8 nC/μF) [7]. Thus, the amount of Q max measured in dysgenic myotubes expressing Ca v 1.1 AA (5.9 ± 0.5 nC/μF) is in good agreement with the previously published values, and indicates that the IQ/AA mutation may have also disrupted retrograde signaling between Ca v 1.1 and RyR1. The similar expression patterns and comparable colocalization of YFP-Ca v 1.1 and YFP-Ca v 1.1 AA with RyR1 in dysgenic myotubes obtained by confocal microscopy and immunocytochemistry further support the argument that YFP-Ca v 1.1 AA seems to be folded and targeted properly to the T-SR junctions. In addition, much more drastic alternation or deletions in Ca v 1.1 sequence did not have such dramatic effects [12,13].
Third, the IQ/AA mutation in C-terminus of Ca v 1.1 had a dramatic effect on its function as a voltage sensor for EC coupling. Even though amount of Q max in dysgenic myotubes expressing YFP-Ca v 1.1 AA is sufficient to support EC coupling (see above), the voltage-gated Ca 2+ release from SR was completely abolished in these cells. This finding suggests that either tethering of CaM to Ca v 1.1 as a structural subunit or the IQ motif itself is necessary for orthograde signaling between Ca v 1.1 and RyR1 (EC coupling). Overexpression of CaM wt and CaM 1234 in normal myotubes did not significantly affect the peak fluorescence-voltage relationship (ΔF/F-V ) in comparison with uninjected normal myotubes, suggesting that the Ca 2+ -binding ability of CaM does not play a role in skeletal muscle EC coupling in single twitch contractions.
For the first time, the present study shows that the IQ motif plays a role in both orthograde (skeletal muscle EC coupling) and retrograde (Ca 2+ current) signaling between Ca v 1.1 and RyR1 in vivo. Several regions of RyR1 were shown to participate in protein-protein interactions between Ca v 1.1 and RyR1. However, until recently only the II-III loop of the Ca v 1.1 has been thought to be necessary to convey orthograde and retrograde signaling between Ca v 1.1 and RyR1. The present findings suggest that the C-terminus in addition to the II-III loop participates in and is necessary for the correct transmission of signals between Ca v 1.1 and RyR1. These results support previously published in vivo findings that in addition to the II-III loop of Ca v 1.1 additional intracellular loops of Ca v 1.1 are necessary to restore the full extent of orthograde and retrograde signaling between Ca v 1.1 and RyR1 [15]. The present findings also support in vitro results from pull-down assays, where it was demonstrated that CaM-binding region of RyR1 (3614-3543) interacts with the proximal C-terminus of Ca v 1.1 (1393-1527) in the absence of CaM [18,19]. It was also shown that CaM binding to the RyR1 is not essential for skeletal EC coupling [20]. This would indicate together with binding studies [18] that CaM association to either Ca v 1.1 or RyR1 is not crucial for skeletal muscle EC coupling, but CaM-binding domains of both Ca v 1.1 and RyR1 are. For example, it has been shown that CaM-binding region of RyR1 binds to IQ peptide of Ca v 1.2 and in pull-down assay binds to Ca v 1.1 [18]. It still remains to be determined whether CaM itself needs to be tethered to Ca v 1.1 to ensure signaling and more experiments are in progress.
In conclusion, the results from confocal microscopy, immunocytochemistry, charge movement, and Ca 2+ transients obtained from dysgenic myotubes expressing YFP-Ca v 1.1 AA indicate that the IQ motif in the C-terminus of Ca v 1.1 plays a crucial role in both orthograde (EC coupling) and retrograde (Ca 2+ current) signaling between Ca v 1.1 and RyR1.