The IQ Motif is Crucial for Ca _v 1.1 Function

Abstract

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 Ca_v1.1 also exhibits Ca²⁺-/CaM-dependent modulation. Here, whole-cell Ca²⁺ current, Ca²⁺ transient, and maximal, immobilization-resistant charge movement (𝑄_max) recordings were obtained from cultured mouse myotubes, to test a role of IQ motif in function of Ca_v1.1. The effect of introducing mutation (IQ to AA) of IQ motif into Ca_v1.1 was examined. In dysgenic myotubes expressing YFP-Ca_v1.1_AA, 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 𝑄_max in dysgenic myotubes expressing YFP-Ca_v1.1_AA was similar to that of normal myotubes. These findings suggest that the IQ motif of the Ca_v1.1 may be an unrecognized site of structural and functional coupling between DHPR and RyR.

1. Introduction

Calcium entering the cell through voltage-gated Ca²⁺ 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 Ca²⁺-dependent modulation of channel activity mediated by the Ca²⁺-binding protein calmodulin (CaM) is found in many ion channels including the Ca_v1 family [1]. Ca²⁺-dependent inactivation (CDI) of Ca_v1.2 is mediated by CaM, and its structural determinants have been assigned to the proximal region of the C-terminus of Ca_v1.2 [1, 2]. Three domains have been identified within this region: a Ca²⁺ binding EF-hand motif, a CaM-tethering site, and a CaM-binding IQ motif. The EF-hand 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 preIQ₃ and IQ motifs, resides 50 amino acids downstream from the EF-hand motif and binds Ca²⁺-free CaM (apo-CaM) at resting [Ca²⁺]_i. The IQ motif resides downstream from the EF-hand motif and the pre-IQ₃ domain, and it binds Ca²⁺-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 Ca²⁺ channel (Ca_v1.1) also displays CDI mediated by CaM and that CaM associates with Ca_v1.1 in vivo [3]. The initial 200 amino acids of the C-terminus of the Ca_v1.1 are highly conserved and contain the above-described domains including the IQ motif. CaM binding to the IQ motif of Ca_v1.2 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_v1.2 resulted in ablation of CDI and significant reduction of apoCaM binding to Ca_v1.2 [1, 2, 4]. Whether the IQ motif in Ca_v1.1 plays a similar role remains to be determined.

In the present work, myotubes cultured from normal and dysgenic (lacking endogenous Ca_v1.1) mice were used to investigate the role of the IQ motif in the function of Ca_v1.1. The results presented demonstrate that the IQ motif in the C-terminus of Ca_v1.1 is critical for function of Ca_v1.1 as a voltage sensor as well as Ca²⁺ channel. Furthermore, the results indicate that the IQ motif may be a previously unrecognized site of protein-protein interaction between Ca_v1.1 and the skeletal muscle ryanodine receptor (RyR1) and may play a role in skeletal muscle excitation-contraction (EC) coupling.

2. Experimental Procedures

2.1. Molecular Biology

The coding sequence of yellow fluorescent protein- (YFP-) tagged Ca_v1.1 channel (YFP-Ca_v1.1) 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_v1.1 [6] were substituted with alanine (A) using the QuikChange II mutagenesis kit (Stratagene, La Jolla, CA), using the YFP-Ca_v1.1 as a template. The construct YFP-Ca_v1.1_AA was verified by restriction digest analysis and sequencing.

2.2. 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_v1.1 or YFP-Ca_v1.1_AA at concentrations of 0.2 μg/μL, respectively. In experiments assessing the effects of CaM on Ca²⁺ transients, normal myotubes (~one week in culture) were injected with expression plasmids encoding CaM_wt or CaM₁₂₃₄ (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.

2.3. 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 Cs₂-EGTA, 5 MgCl₂, and 10 HEPES (pH 7.4 with CsOH). The external solution contained (in mM) 145 tetraethylammonium chloride (TEA-Cl), 10 CaCl₂, 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 Ca²⁺ 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. Ca²⁺ currents were normalized by linear cell capacitance (expressed in pA/pF). Values for 𝐺_max, the maximal Ca²⁺ conductance, were obtained by fitting the measured currents according to the following equation:𝐼_peak=𝐺_max𝑉−𝑉_𝑅1+exp−𝑉−𝑉_1/2/𝑘, (1) 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 Ca²⁺ conductance, and 𝑘 is a slope factor.

The fraction of current remaining at the end of an 800 ms test pulse (𝑟₈₀₀) 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 𝑟₈₀₀=𝐼_end𝐼_peak. (2)

For measurements of charge movement, 0.5 mM Cd²⁺ and 0.1 mM La³⁺ were added to the external solution to block Ca²⁺ 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 Ca²⁺ release from the SR, the Ca²⁺ indicator K₅-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:Δ𝐹𝐹=(Δ𝐹/𝐹)_max1+exp𝑉_𝐹1/2−𝑉/𝑘_𝐹, (3) where (Δ𝐹/𝐹)_max is the maximal fluorescent change, 𝑉_𝐹1/2 is the potential for half-maximal activation of the Ca²⁺ 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%.

2.4. Confocal Microscopy

Cells were bathed in rodent ringer (in mM: 146 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 11 glucose, 10 HEPES; pH 7.4 adjusted with NaOH) and examined with an LSM 510 META laser scanning microscope (Zeiss, Thornwood, New York) with 40X oil immersion objective. The laser line (514 nm) of the argon laser (30 mW maximum output, operated at 50% or 6.3 A) was used to excite YFP 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_v1.1 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 (phosphate-buffered 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.

3. Results

Ca²⁺-binding ability of CaM does not affect skeletal muscle EC coupling. First, I addressed the question whether the Ca²⁺-binding ability of CaM plays any role in skeletal muscle EC coupling. Overexpressed mutant CaM which does not bind Ca²⁺ (CaM₁₂₃₄) can displace approximately 70% of endogenous CaM, as reflected by abolishment of CDI of Ca_v1.1 [3]. However, overexpression of either CaM_wt or CaM₁₂₃₄ in normal myotubes did not significantly affect either current-voltage (𝐼/𝑉) relationship (Figure 1(a)) or voltage-gated Ca²⁺ 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 Ca²⁺-binding ability of CaM or CaM itself does not play a role in skeletal muscle EC coupling. However, CaM associates with Ca_v1.1 in vivo [3] and that indicates the possibility that CaM may still serve as a structural subunit of Ca_v1.1, that is, that interaction between CaM and Ca_v1.1 can stabilize the DHPR complex. By doing so, it may also ensure proper structural and functional coupling between DHPR and RyR1.

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(b)

Figure 1 

Ca2+-binding ability of CaM does not affect skeletal muscle EC coupling. (a) The average peak current density (𝐼peak) is plotted as a function of membrane potential (𝑉test). Data were obtained from the indicated number of myotubes for each group. The smooth lines through the data were generated by using equation (1) (see Methods) and the average values. (b) The average peak fluorescence (Δ𝐹/𝐹) is plotted as a function of membrane potential (𝑉test). Data were obtained from the indicated number of myotubes for each group. The smooth lines through data represent Boltzmann fits to the average data using equation (3) (see Methods).

Therefore, I examined whether CaM association with Ca_v1.1 is necessary for its function as a voltage sensor for EC coupling. The IQ motif of Ca_v1.1 has been shown to bind CaM similar to IQ motifs of Ca_v1.3 and Ca_v2 channels [7]. Introduction of the mutation IQ/AA in the IQ motif of the cardiac L-type Ca²⁺ channel (Ca_v1.2) resulted in abolishment of CDI and significant reduction of apoCaM binding to Ca_v1.2 [2, 4]. Thus, corresponding IQ motif mutation in the C-terminus of Ca_v1.1 was obvious place to start.

The mutation (IQ/AA) in the CaM-binding site of Ca_v1.1 disables function of Ca_v1.1 as a Ca²⁺ channel and voltage sensor for EC coupling. I introduced the IQ/AA mutation in the C-terminus of Ca_v1.1 and investigated how this mutation will alter Ca_v1.1 function as Ca²⁺ channel and voltage sensor for EC coupling. Introduction of the mutation IQ/AA in the IQ motif of Ca_v1.2 resulted in abolishment of CDI and significant reduction of apoCaM binding to Ca_v1.2 [2, 4]. Whether IQ motif in the Ca_v1.1 plays a similar role is unknown. Dysgenic myotubes expressing either YFP-Ca_v1.1 or YFP-Ca_v1.1_AA were used to examine the role of IQ motif in Ca²⁺-dependent inactivation (CDI) of Ca_v1.1. Injections of plasmids encoding various constructs of Ca_v1.1 into dysgenic myotubes at concentrations of 0.2–0.5 μg/μL have been previously demonstrated to produce a similar extent of maximal, immobilization-resistant charge movement and similar Ca²⁺ current densities as normal myotubes, which corresponds to similar protein expression levels [3, 6, 8–10].

Figure 2(a) shows Ca²⁺ currents mediated by YFP-Ca_v1.1 expressed in dysgenic myotube. The fraction of current remaining at the end of the pulse (𝑟₈₀₀) displayed a U-shaped 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 𝑟₈₀₀ value of 0.9 ± 0.08 (𝑛=7), and maximal at a test potential of +40 mV, as reflected by a minimum 𝑟₈₀₀ value of 0.74 ± 0.03 (𝑛=7). Correspondingly, the Ca²⁺ current attained its maximum conductance at +40 mV (Figure 1(c)). Thus, Ca²⁺ currents mediated by YFP-Ca_v1.1 displayed a current-dependent inactivation process, current-voltage (𝐼-𝑉) relationship (Figure 2(c)), and maximal Ca²⁺ ion conductance (𝐺_max=166±18 nS/nF; 𝑛=7) similar to the endogenous Ca_v1.1 of normal myotubes [3]. These results suggest that YFP fused to the N-terminus of Ca_v1.1 does not interfere with channel function.

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(b)

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(d)

Figure 2 

The IQ/AA mutation disables function of Cav1.1 as a Ca2+ channel and voltage sensor for EC coupling. Representative whole-cell L-type Ca2+ currents recorded from dysgenic myotubes expressing either (a) YFP-Cav1.1 (cell 1 (05-10-02); linear capacitance 𝐶=587 pF) or (b) YFP-Cav1.1AA (cell 1 (01-13-03); linear capacitance 𝐶=201 pF). Note that the current scales are different in (a) and (b). (c) The average peak current density (𝐼peak) is plotted as a function of membrane potential (𝑉test). Data were obtained from the indicated number of myotubes for each group. The smooth lines through the data were generated by using equation (1) (see Methods) and the average values. (d) The average peak fluorescence (Δ𝐹/𝐹) is plotted as a function of membrane potential (𝑉test). Data were obtained from the indicated number of myotubes for each group. The smooth lines through data represent Boltzmann fits to the average data using equation (3) (see Methods).

In contrast, dysgenic myotubes expressing YFP-Ca_v1.1_AA (Figures 2(b) and 2(c)) displayed either very small (<1pA/pF) or no measurable Ca²⁺ currents. This is a very dramatic and surprising result considering that the corresponding mutation (IQ/AA) in the IQ motif of Ca_v1.2 resulted only in ablation of CDI but did not affect the 𝐼-𝑉 relationship of Ca²⁺ currents mediated by Ca_v1.2 [2]. Further voltage-gated Ca²⁺ currents and SR Ca²⁺ release were measured simultaneously from dysgenic myotubes expressing YFP-Ca_v1.1_AA and compared with recordings from uninjected normal myotubes and normal myotubes overexpressing CaM_wt or CaM₁₂₃₄. The voltage-gated Ca²⁺ release from SR was completely abolished in dysgenic myotubes expressing YFP-Ca_v1.1_AA (Figure 2(d)).

The loss of Ca_v1.1_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_v1.1 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_v1.1 is necessary for orthograde signaling from Ca_v1.1 to RyR1, which underlies skeletal muscle EC coupling.

The IQ/AA mutation does not prevent proper targeting of Ca_v1.1 into sarcolemma. The severe loss of function, abolished Ca²⁺ current and orthograde signaling mediated by the Ca_v1.1_AA, could be explained by compromised targeting of Ca_v1.1 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_v1.1 or YFP-Ca_v1.1_AA. Expression of YFP-Ca_v1.1 (a) or YFP-Ca_v1.1_AA (b) resulted in the appearance of small yellow fluorescence puncta located near the cell surface. The small puncta correspond to groups of Ca_v1.1 localized to T-SR junctions; these puncta are similar in size and distribution to those of Ca_v1.1 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.

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(b)

Figure 3 

YFP-Cav1.1AA displays similar expression pattern as YFP-Cav1.1 in dysgenic myotubes. Confocal images of either YFP-Cav1.1 (a) or YFP-Cav1.1AA (b) yellow fluorescence in dysgenic myotubes. Bar 50 μm.

To confirm targeting of YFP-Ca_v1.1_AA to the sarcolemma, the 𝑄_max was measured at +40 mV (Figure 4). The 𝑄_max in dysgenic myotubes expressing Ca_v1.1_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_v1.1 did not prevent the protein from being properly targeted or undergoing voltage-dependent conformational changes, which strongly suggest proper folding as intramembrane segment S4 of Ca_v1.1 is responsible for voltage-dependent movement.

Figure 4 

Cav1.1AA generates normal densities of intramembrane charge movement. The average maximal, immobilization-resistant charge movement at +40 mV (𝑄max) obtained from the indicated number of myotubes for each group. The charge movements were elicited by 25 ms depolarizations from a pedestal potential (−50 mV) to +40 mV. Symbols and error bars represent mean ± SEM.

To further confirm Ca_v1.1_AA proper targeting into T-SR junctions and site of EC coupling, I investigated colocalization of Ca_v1.1 and RyR1. Dysgenic myotubes expressing either YFP-Ca_v1.1 or YFP-Ca_v1.1_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_v1.1 and RyR1 in T-SR junctions in vivo (see Figures 5(g) and 5(h)). Colocalization patterns of YFP-Ca_v1.1_AA with RyR1 were compared to YFP-Ca_v1.1 and RyR1 patterns in 5 different experiments. Colocalization patterns of either YFP-Ca_v1.1 or YFP-Ca_v1.1_AA with RyR1 were similar and have been analyzed by MetaMorph 7 software (Molecular Devices). Colocalization in near surface slices of z-stacks of YFP-Ca_v1.1 and RyR1 was 83±4% (𝑛=7), and colocalization of YFP-Ca_v1.1_AA and RyR1 was 85±2% (𝑛=7). These results strongly suggest that YFP-Ca_v1.1_AA is targeted into T-SR junctions.

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Figure 5 

Cav1.1AA is targeted to T-SR junctions. Confocal images of colocalization of either YFP-Cav1.1 (a) or YFP-Cav1.1AA (b) green fluorescence and immunolabeled RyR1 ((d) and (e)) red fluorescence in dysgenic myotubes. (g) and (h) represent overlay of (a) and (d), and (b) and (e), respectively. The control dysgenic myotube (no Cav1.1) immunolabeled without (c) and with (f) primary Ab(34C). Bar, 20 μm.

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_v1.1 sequence did not have such dramatic effects [12, 13].

Taking altogether, the loss of both ionic Ca²⁺ current and skeletal muscle EC coupling in Ca_v1.1_AA along with charge movement similar to normal myotubes suggests that the IQ motif of the Ca_v1.1 may be unrecognized site of protein-protein interaction between Ca_v1.1 and RyR1 and play a role in both orthograde and retrograde signaling.

4. Discussion

The present study provides new information about the skeletal muscle L-type Ca²⁺ channel (Ca_v1.1). Specifically, the data demonstrate in vivo that the IQ motif in the C-terminus of Ca_v1.1 is critical for function of Ca_v1.1 as a voltage sensor as well as a Ca²⁺ 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_v1.1 and RyR1 and, thus, may play a role in skeletal muscle EC coupling.

Ca_v1.1 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_v1.1 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_v1.1 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_v1.1 to RyR1, in which movement of the voltage sensors in Ca_v1.1 trigger opening of RyR1 and release of Ca²⁺ from the SR (EC coupling). Retrograde signaling is communication from RyR1 to Ca_v1.1, in which RyR1 somehow increases the amount of L-type Ca²⁺ current mediated by Ca_v1.1 [8, 9].

The Ca²⁺ conductance of Ca_v1.1 channel is not necessary for functional excitation-contraction coupling between RyR1 and Ca_v1.1; however, a direct protein-protein interaction between these two proteins in multiple sites is. It has been shown that cytoplasmic loops of Ca_v1.1 and several regions of RyR1 play important role for normal physiological EC coupling in skeletal muscle [10, 14–17]. It has been also shown that protein-protein interaction between RyR1 and Ca_v1.1 is necessary for Ca_v1.1 display of Ca²⁺ conductance (retrograde signaling) [8]. It is clear that there are multiple contact sites between RyR1 and Ca_v1.1 and not all of them are recognized and understood, yet. The most investigated region of contact between RyR1 and Ca_v1.1 in Ca_v1.1 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_v1.1 or YFP-Ca_v1.1_AA were used to examine the role of the IQ motif in both functions of Ca_v1.1, as a voltage sensor in EC coupling and Ca²⁺ channel. The primary cultures of skeletal muscle myotubes provide a natural cellular environment for Ca_v1.1. First, I examined whether a fusion of YFP to Ca_v1.1 would interfere with its function. The Ca²⁺ currents mediated by YFP-Ca_v1.1 displayed an 𝐼-𝑉 relationship similar to the endogenous Ca_v1.1 [3], suggesting that YFP fused on the N-terminus of Ca_v1.1 does not interfere with its channel function, as was also shown by others [5]. Endogenous Ca_v1.1 also exhibits CaM-mediated Ca²⁺-dependent inactivation (CDI) [3]. The Ca²⁺ currents mediated by YFP-Ca_v1.1 also displayed current-dependent inactivation similar to the CDI of endogenous Ca_v1.1, further supporting observation that fusion of YFP with Ca_v1.1 does not interfere with channel function.

Second, I examined how IQ/AA mutation in Ca_v1.1 will affect its function. Surprisingly, the intriguing finding of the present study was that dysgenic myotubes expressing YFP-Ca_v1.1_AA displayed either very small or no measurable Ca²⁺ currents. Significant decrease or abolishment of Ca²⁺ current through Ca_v1.1 could have resulted from improper targeting or folding of the protein. If Ca_v1.1_AA was retained inside of myotubes due to incorrect folding and targeting, neither Ca²⁺ currents nor 𝑄_max would be obtained. The absence of Ca²⁺ currents in some of the dysgenic myotubes expressing YFP-Ca_v1.1_AA would suggest both. However, even though the Ca²⁺ current was absent, 𝑄_max comparable with normal myotubes was observed. The 𝑄_max measured in dysgenic myotubes expressing Ca_v1.1_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_v1.1 at the similar experimental conditions elsewhere [6, 7].

The amount of 𝑄_max in dysgenic myotubes expressing YFP-Ca_v1.1_AA suggests that IQ/AA mutation in Ca_v1.1 did not prevent the protein from being properly targeted and that protein can undergo voltage-dependent conformational changes. The size of small measurable Ca²⁺ currents measured in some (6 out of 14) of the dysgenic myotubes expressing Ca_v1.1_AA (<1pA/pF) was similar to L-type Ca²⁺ currents measured in dyspedic (lacking a functional gene of RyR1) myotubes [7], suggesting a loss of retrograde signaling from RyR1. Endogenous Ca_v1.1 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_v1.1_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_v1.1 and RyR1. The similar expression patterns and comparable colocalization of YFP-Ca_v1.1 and YFP-Ca_v1.1_AA with RyR1 in dysgenic myotubes obtained by confocal microscopy and immunocytochemistry further support the argument that YFP-Ca_v1.1_AA seems to be folded and targeted properly to the T-SR junctions. In addition, much more drastic alternation or deletions in Ca_v1.1 sequence did not have such dramatic effects [12, 13].

Third, the IQ/AA mutation in C-terminus of Ca_v1.1 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_v1.1_AA is sufficient to support EC coupling (see above), the voltage-gated Ca²⁺ release from SR was completely abolished in these cells. This finding suggests that either tethering of CaM to Ca_v1.1 as a structural subunit or the IQ motif itself is necessary for orthograde signaling between Ca_v1.1 and RyR1 (EC coupling). Overexpression of CaM_wt and CaM₁₂₃₄ in normal myotubes did not significantly affect the peak fluorescence-voltage relationship (Δ𝐹/𝐹-𝑉) in comparison with uninjected normal myotubes, suggesting that the Ca²⁺-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 (Ca²⁺ current) signaling between Ca_v1.1 and RyR1 in vivo. Several regions of RyR1 were shown to participate in protein-protein interactions between Ca_v1.1 and RyR1. However, until recently only the II-III loop of the Ca_v1.1 has been thought to be necessary to convey orthograde and retrograde signaling between Ca_v1.1 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_v1.1 and RyR1. These results support previously published in vivo findings that in addition to the II-III loop of Ca_v1.1 additional intracellular loops of Ca_v1.1 are necessary to restore the full extent of orthograde and retrograde signaling between Ca_v1.1 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_v1.1 (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_v1.1 or RyR1 is not crucial for skeletal muscle EC coupling, but CaM-binding domains of both Ca_v1.1 and RyR1 are. For example, it has been shown that CaM-binding region of RyR1 binds to IQ peptide of Ca_v1.2 and in pull-down assay binds to Ca_v1.1 [18]. It still remains to be determined whether CaM itself needs to be tethered to Ca_v1.1 to ensure signaling and more experiments are in progress.

In conclusion, the results from confocal microscopy, immunocytochemistry, charge movement, and Ca²⁺ transients obtained from dysgenic myotubes expressing YFP-Ca_v1.1_AA indicate that the IQ motif in the C-terminus of Ca_v1.1 plays a crucial role in both orthograde (EC coupling) and retrograde (Ca²⁺ current) signaling between Ca_v1.1 and RyR1.

Acknowledgments

The authors thanks Dr. David Yue for kindly providing CaM_wt and CaM₁₂₃₄ constructs Dr. Kurt Beam for discussions, providing YFP-Ca_v1.1 construct, and for his support of preliminary studies and Dr. Brett Adams for his helpful insights and constructive criticism of the paper. This work was supported by MDA Research Grant and PIRG06-GA-2009-256580, Marie Curie Actions FP7-PEOPLE-2009-RG, EU.