# The IQ Motif is Crucial for Cav1.1 Function

Authors: Katarina Stroffekova
Journal: Journal of Biomedicine and Biotechnology
 (2011)

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## 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 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.


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## Body

## 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 Cav1 family [1]. Ca2+-dependent inactivation (CDI) of Cav1.2 is mediated by CaM, and its structural determinants have been assigned to the proximal region of the C-terminus of Cav1.2 [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 (Cav1.1) also displays CDI mediated by CaM and that CaM associates with Cav1.1 in vivo [3]. The initial 200 amino acids of the C-terminus of the Cav1.1 are highly conserved and contain the above-described domains including the IQ motif. CaM binding to the IQ motif of Cav1.2 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 Cav1.2 resulted in ablation of CDI and significant reduction of apoCaM binding to Cav1.2 [1, 2, 4]. Whether the IQ motif in Cav1.1 plays a similar role remains to be determined.In the present work, myotubes cultured from normal and dysgenic (lacking endogenous Cav1.1) mice were used to investigate the role of the IQ motif in the function of Cav1.1. The results presented demonstrate that the IQ motif in the C-terminus of Cav1.1 is critical for function of Cav1.1 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 Cav1.1 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 Cav1.1 channel (YFP-Cav1.1) 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 Cav1.1 [6] were substituted with alanine (A) using the QuikChange II mutagenesis kit (Stratagene, La Jolla, CA), using the YFP-Cav1.1 as a template. The construct YFP-Cav1.1AA 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-Cav1.1 or YFP-Cav1.1AA 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 CaMwt 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 Gmax, the maximal Ca2+ conductance, were obtained by fitting the measured currents according to the following equation:(1)Ipeak=Gmax⁡(V-VR){1+exp⁡[-(V-V1/2)/k]},
where Ipeak is the peak current activated at the test potential V, VR is the extrapolated reversal potential, V1/2 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 (2)r800=IendIpeak.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 (Qmax) 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 (Fbase) 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=Ftransient-Fbase), and F is Fbase. Peak fluorescence during each test pulse was plotted as a function of test potential V and fitted according to the following equation:(3)ΔFF=(ΔF/F)max⁡{1+exp⁡[(VF1/2-V)/kF]},
where (ΔF/F)max is the maximal fluorescent change, VF1/2 is the potential for half-maximal activation of the Ca2+ transient, and kF 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 Cav1.1 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.


## 2.1. Molecular Biology

The coding sequence of yellow fluorescent protein- (YFP-) tagged Cav1.1 channel (YFP-Cav1.1) 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 Cav1.1 [6] were substituted with alanine (A) using the QuikChange II mutagenesis kit (Stratagene, La Jolla, CA), using the YFP-Cav1.1 as a template. The construct YFP-Cav1.1AA 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-Cav1.1 or YFP-Cav1.1AA 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 CaMwt 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 Gmax, the maximal Ca2+ conductance, were obtained by fitting the measured currents according to the following equation:(1)Ipeak=Gmax⁡(V-VR){1+exp⁡[-(V-V1/2)/k]},
where Ipeak is the peak current activated at the test potential V, VR is the extrapolated reversal potential, V1/2 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 (2)r800=IendIpeak.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 (Qmax) 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 (Fbase) 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=Ftransient-Fbase), and F is Fbase. Peak fluorescence during each test pulse was plotted as a function of test potential V and fitted according to the following equation:(3)ΔFF=(ΔF/F)max⁡{1+exp⁡[(VF1/2-V)/kF]},
where (ΔF/F)max is the maximal fluorescent change, VF1/2 is the potential for half-maximal activation of the Ca2+ transient, and kF 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 Cav1.1 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 Cav1.1 [3]. However, overexpression of either CaMwt 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 Cav1.1 in vivo [3] and that indicates the possibility that CaM may still serve as a structural subunit of Cav1.1, that is, that interaction between CaM and Cav1.1 can stabilize the DHPR complex. By doing so, it may also ensure proper structural and functional coupling between DHPR and RyR1.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).
(a)(b)Therefore, I examined whether CaM association with Cav1.1 is necessary for its function as a voltage sensor for EC coupling. The IQ motif of Cav1.1 has been shown to bind CaM similar to IQ motifs of Cav1.3 and Cav2 channels [7]. Introduction of the mutation IQ/AA in the IQ motif of the cardiac L-type Ca2+ channel (Cav1.2) resulted in abolishment of CDI and significant reduction of apoCaM binding to Cav1.2 [2, 4]. Thus, corresponding IQ motif mutation in the C-terminus of Cav1.1 was obvious place to start.The mutation (IQ/AA) in the CaM-binding site of Cav1.1 disables function of Cav1.1 as a Ca2+ channel and voltage sensor for EC coupling. I introduced the IQ/AA mutation in the C-terminus of Cav1.1 and investigated how this mutation will alter Cav1.1 function as Ca2+ channel and voltage sensor for EC coupling. Introduction of the mutation IQ/AA in the IQ motif of Cav1.2 resulted in abolishment of CDI and significant reduction of apoCaM binding to Cav1.2 [2, 4]. Whether IQ motif in the Cav1.1 plays a similar role is unknown. Dysgenic myotubes expressing either YFP-Cav1.1 or YFP-Cav1.1AA were used to examine the role of IQ motif in Ca2+-dependent inactivation (CDI) of Cav1.1. Injections of plasmids encoding various constructs of Cav1.1 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].Figure2(a) shows Ca2+ currents mediated by YFP-Cav1.1 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-Cav1.1 displayed a current-dependent inactivation process, current-voltage (I-V) relationship (Figure 2(c)), and maximal Ca2+ ion conductance (Gmax=166±18 nS/nF; n=7) similar to the endogenous Cav1.1 of normal myotubes [3]. These results suggest that YFP fused to the N-terminus of Cav1.1 does not interfere with channel function.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).
(a)(b)(c)(d)In contrast, dysgenic myotubes expressing YFP-Cav1.1AA (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 Cav1.2 resulted only in ablation of CDI but did not affect the I-V relationship of Ca2+ currents mediated by Cav1.2 [2]. Further voltage-gated Ca2+ currents and SR Ca2+ release were measured simultaneously from dysgenic myotubes expressing YFP-Cav1.1AA  and compared with recordings from uninjected normal myotubes and normal myotubes overexpressing CaMwt or CaM1234. The voltage-gated Ca2+ release from SR was completely abolished in dysgenic myotubes expressing YFP-Cav1.1AA (Figure 2(d)).The loss of Cav1.1AA 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 Cav1.1 was significantly disturbed. If the latter possibility is the case, this result suggests that either the IQ motif itself or association of CaM with Cav1.1 is necessary for orthograde signaling from Cav1.1 to RyR1, which underlies skeletal muscle EC coupling.The IQ/AA mutation does not prevent proper targeting of Cav1.1 into sarcolemma. The severe loss of function, abolished Ca2+ current and orthograde signaling mediated by the Cav1.1AA, could be explained by compromised targeting of Cav1.1 to the T-SR junction as a result of incomplete protein folding.Figure3 shows confocal images of yellow fluorescence from a dysgenic myotube expressing either YFP-Cav1.1 or YFP-Cav1.1AA. Expression of YFP-Cav1.1 (a) or YFP-Cav1.1AA (b) resulted in the appearance of small yellow fluorescence puncta located near the cell surface. The small puncta correspond to groups of Cav1.1 localized to T-SR junctions; these puncta are similar in size and distribution to those of Cav1.1 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.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. Bar50 μm.
(a)(b)To confirm targeting of YFP-Cav1.1AA to the sarcolemma, the Qmax was measured at +40 mV (Figure 4). The Qmax in dysgenic myotubes expressing Cav1.1AA (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 Cav1.1 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 Cav1.1 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 Cav1.1AA proper targeting into T-SR junctions and site of EC coupling, I investigated colocalization of Cav1.1 and RyR1. Dysgenic myotubes expressing either YFP-Cav1.1 or YFP-Cav1.1AA (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 (redfluorescence). Colocalization of green and red fluorescence results in yellow pattern suggests colocalization of YFP-Cav1.1 and RyR1 in T-SR junctions in vivo (see Figures 5(g) and 5(h)). Colocalization patterns of YFP-Cav1.1AA with RyR1 were compared to YFP-Cav1.1 and RyR1 patterns in 5 different experiments. Colocalization patterns of either YFP-Cav1.1 or YFP-Cav1.1AA with RyR1 were similar and have been analyzed by MetaMorph 7 software (Molecular Devices). Colocalization in near surface slices of z-stacks of YFP-Cav1.1 and RyR1 was 83±4% (n=7), and colocalization of YFP-Cav1.1AA and RyR1 was 85±2% (n=7). These results strongly suggest that YFP-Cav1.1AA is targeted into T-SR junctions.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.
(a)(b)(c)(d)(e)(f)(g)(h)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 Cav1.1 sequence did not have such dramatic effects [12, 13].Taking altogether, the loss of both ionic Ca2+ current and skeletal muscle EC coupling in Cav1.1AA along with charge movement similar to normal myotubes suggests that the IQ motif of the Cav1.1 may be unrecognized site of protein-protein interaction between Cav1.1 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 (Cav1.1). Specifically, the data demonstrate in vivo that the IQ motif in the C-terminus of Cav1.1 is critical for function of Cav1.1 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 Cav1.1 and RyR1 and, thus, may play a role in skeletal muscle EC coupling.Cav1.1 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 Cav1.1 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 Cav1.1 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 Cav1.1 to RyR1, in which movement of the voltage sensors in Cav1.1 trigger opening of RyR1 and release of Ca2+ from the SR (EC coupling). Retrograde signaling is communication from RyR1 to Cav1.1, in which RyR1 somehow increases the amount of L-type Ca2+ current mediated by Cav1.1 [8, 9].The Ca2+ conductance of Cav1.1 channel is not necessary for functional excitation-contraction coupling between RyR1 and Cav1.1; however, a direct protein-protein interaction between these two proteins in multiple sites is. It has been shown that cytoplasmic loops of Cav1.1 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 Cav1.1 is necessary for Cav1.1 display of Ca2+ conductance (retrograde signaling) [8]. It is clear that there are multiple contact sites between RyR1 and Cav1.1 and not all of them are recognized and understood, yet. The most investigated region of contact between RyR1 and Cav1.1 in Cav1.1 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-Cav1.1 or YFP-Cav1.1AA were used to examine the role of the IQ motif in both functions of Cav1.1, as a voltage sensor in EC coupling and Ca2+ channel. The primary cultures of skeletal muscle myotubes provide a natural cellular environment for Cav1.1. First, I examined whether a fusion of YFP to Cav1.1 would interfere with its function. The Ca2+ currents mediated by YFP-Cav1.1 displayed an I-V relationship similar to the endogenous Cav1.1 [3], suggesting that YFP fused on the N-terminus of Cav1.1 does not interfere with its channel function, as was also shown by others [5]. Endogenous Cav1.1 also exhibits CaM-mediated Ca2+-dependent inactivation (CDI) [3]. The Ca2+ currents mediated by YFP-Cav1.1 also displayed current-dependent inactivation similar to the CDI of endogenous Cav1.1, further supporting observation that fusion of YFP with Cav1.1 does not interfere with channel function.Second, I examined how IQ/AA mutation in Cav1.1 will affect its function. Surprisingly, the intriguing finding of the present study was that dysgenic myotubes expressing YFP-Cav1.1AA displayed either very small or no measurable Ca2+ currents. Significant decrease or abolishment of Ca2+ current through Cav1.1 could have resulted from improper targeting or folding of the protein. If Cav1.1AA was retained inside of myotubes due to incorrect folding and targeting, neither Ca2+ currents nor Qmax would be obtained. The absence of Ca2+ currents in some of the dysgenic myotubes expressing YFP-Cav1.1AA would suggest both. However, even though the Ca2+ current was absent, Qmax comparable with normal myotubes was observed. The Qmax measured in dysgenic myotubes expressing Cav1.1AA was significantly larger (P<0.001) than in dysgenic myotubes alone, but similar to that of normal myotubes measured here and to the Qmax measured in dysgenic myotubes expressing various constructs of wt Cav1.1 at the similar experimental conditions elsewhere [6, 7].The amount ofQmax in dysgenic myotubes expressing YFP-Cav1.1AA suggests that IQ/AA mutation in Cav1.1 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 Cav1.1AA (<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 Cav1.1 channels are present in sarcolemma of the dyspedic myotubes in similar density as in normal myotubes, as was demonstrated by comparable Qmax (dyspedic: 4.0±1.4 nC/μF; normal: 6.4±2.8 nC/μF) [7]. Thus, the amount of Qmax measured in dysgenic myotubes expressing Cav1.1AA (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 Cav1.1 and RyR1. The similar expression patterns and comparable colocalization of YFP-Cav1.1 and YFP-Cav1.1AA with RyR1 in dysgenic myotubes obtained by confocal microscopy and immunocytochemistry further support the argument that YFP-Cav1.1AA seems to be folded and targeted properly to the T-SR junctions. In addition, much more drastic alternation or deletions in Cav1.1 sequence did not have such dramatic effects [12, 13].Third, the IQ/AA mutation in C-terminus of Cav1.1 had a dramatic effect on its function as a voltage sensor for EC coupling. Even though amount of Qmax in dysgenic myotubes expressing YFP-Cav1.1AA 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 Cav1.1 as a structural subunit or the IQ motif itself is necessary for orthograde signaling between Cav1.1 and RyR1 (EC coupling). Overexpression of CaMwt 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 bothorthograde (skeletal muscle EC coupling) and retrograde (Ca2+ current) signaling between Cav1.1 and RyR1 in vivo. Several regions of RyR1 were shown to participate in protein-protein interactions between Cav1.1 and RyR1. However, until recently only the II-III loop of the Cav1.1 has been thought to be necessary to convey orthograde and retrograde signaling between Cav1.1 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 Cav1.1 and RyR1. These results support previously published in vivo findings that in addition to the II-III loop of Cav1.1 additional intracellular loops of Cav1.1 are necessary to restore the full extent of orthograde and retrograde signaling between Cav1.1 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 Cav1.1 (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 Cav1.1 or RyR1 is not crucial for skeletal muscle EC coupling, but CaM-binding domains of both Cav1.1 and RyR1 are. For example, it has been shown that CaM-binding region of RyR1 binds to IQ peptide of Cav1.2 and in pull-down assay binds to Cav1.1 [18]. It still remains to be determined whether CaM itself needs to be tethered to Cav1.1 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-Cav1.1AA indicate that the IQ motif in the C-terminus of Cav1.1 plays a crucial role in both orthograde (EC coupling) and retrograde (Ca2+ current) signaling between Cav1.1 and RyR1.



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*Source: 504649-2011-11-13.xml*