Calmodulin-dependent gating of Ca(v)1.2 calcium channels in the absence of Ca(v)beta subunits - PubMed (original) (raw)

Calmodulin-dependent gating of Ca(v)1.2 calcium channels in the absence of Ca(v)beta subunits

Arippa Ravindran et al. Proc Natl Acad Sci U S A. 2008.

Abstract

It is generally accepted that to generate calcium currents in response to depolarization, Ca(v)1.2 calcium channels require association of the pore-forming alpha(1C) subunit with accessory Ca(v)beta and alpha(2)delta subunits. A single calmodulin (CaM) molecule is tethered to the C-terminal alpha(1C)-LA/IQ region and mediates Ca2+-dependent inactivation of the channel. Ca(v)beta subunits are stably associated with the alpha(1C)-interaction domain site of the cytoplasmic linker between internal repeats I and II and also interact dynamically, in a Ca2+-dependent manner, with the alpha(1C)-IQ region. Here, we describe a surprising discovery that coexpression of exogenous CaM (CaM(ex)) with alpha(1C)/alpha(2)delta in COS1 cells in the absence of Ca(v)beta subunits stimulates the plasma membrane targeting of alpha(1C), facilitates calcium channel gating, and supports Ca2+-dependent inactivation. Neither real-time PCR with primers complementary to monkey Ca(v)beta subunits nor coimmunoprecipitation analysis with exogenous alpha(1C) revealed an induction of endogenous Ca(v)beta subunits that could be linked to the effect of CaM(ex). Coexpression of a calcium-insensitive CaM mutant CaM(1234) also facilitated gating of Ca(v)beta-free Ca(v)1.2 channels but did not support Ca2+-dependent inactivation. Our results show there is a functional matchup between CaM(ex) and Ca(v)beta subunits that, in the absence of Ca(v)beta, renders Ca2+ channel gating facilitated by CaM molecules other than the one tethered to LA/IQ to support Ca2+-dependent inactivation. Thus, coexpression of CaM(ex) creates conditions when the channel gating, voltage- and Ca2+-dependent inactivation, and plasma-membrane targeting occur in the absence of Ca(v)beta. We suggest that CaM(ex) affects specific Ca(v)beta-free conformations of the channel that are not available to endogenous CaM.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Effects of CaMex on the properties of the Cav1.2 channel. The EYFPN-α1C, α2δ and β2d subunits were expressed in COS1 cells in the presence (A) or absence (B) of ECFPN-CaM. Shown are representative traces of _I_Ca recorded in response to 600-ms steps to indicated test potentials (_V_t) from the holding potential _V_h = −90 mV. (a and b) Epifluorescent images of the expressing cells showing distribution of EYFPN-α1C and ECFPN-CaM and obtained with the YFP and CFP filters, respectively. (Scale bars, 4 μm.) Arrows point to PM targeting of EYFPN-α1C. Inactivation time constants (τ) were determined from the fitting of _I_Ca decay by an exponential function: I(t) = I_∞ + I × exp(−_t/τ), where _I_∞ is the steady-state amplitude of the current, I is apparent inactivating component of the initial current. _I_o is the sustained current component determined as the ratio of steady state to peak current amplitudes. (C) Voltage dependence of the sustained component _I_o of the _I_Ca inactivation. (D) The averaged I–V curves for _I_Ca recorded in the absence (filled circles) or presence of coexpressed CaM (open circles). Currents were measured with 30-s intervals between 0.6-s test pulses in the range of −60 to +80 mV applied with 10-mV increments from _V_h = −90 mV. Smooth lines represent fitting by equation _I_Ca = _G_max (V − _E_rev)/(1 + exp[(V − _V_0.5)/_k_I–V]), where _G_max is maximum conductance, _E_rev is the approximated reversal potential, _V_0.5 is voltage at 50% of _I_Ca activation, and _k_I–V is slope factor. Cav1.2: _V_0.5 = 14.6 ± 0.6, _k_I–V = −9.9 ± 0.3, _E_rev = 105.2 ± 3.5 mV (n = 5); Cav1.2 + CaMex: _V_0.5 = 2.4 ± 0.6, _k_I–V = −5.0 ± 0.5, _E_rev = 93.9 ± 1.3 mV (n = 8). (E) Voltage dependence of τ for _I_Ca recorded in the absence (filled circles) or presence of CaMex (open circles). All error bars reflect SEM. (F) Inhibition of _I_Ca through the EYFPN-α1C/α2δ/β2d/CaMex channel by 2 μM (+)PN200–110. _V_h = −90 mV, _V_t = +30 mV.

Fig. 2.

Fig. 2.

Effects of CaMex on the properties of the β-deficient Cav1.2 channel. (A) Ca2+ channel activity in COS1 cells expressing EYFPN-α1C and CaMex (a), α2δ (b), β2d (c), or β2d +CaMex (d). Shown are representative traces (n = 5–10) of maximal _I_Ca evoked by 600-ms test pulses to +20 mV (a and c), +30 mV (b), or +50 mV (d) applied from _V_h = −90 mV. (B) Relative distribution of EYFPN-α1C in PM over the cytoplasm in the presence of CaMex (1), α2δ (2), β2d (3), β2d + CaMex (4), α2δ + β2d (5), α2δ + CaMex (6), or α2δ + β2d + CaMex (7). The ratio of fluorescence intensity in PM over the area underneath PM was averaged after background subtraction in each cell. The ratio <1.0 indicates lack of significant α1C PM targeting. ANOVA statistical analysis with Tukey–Kramer multiple comparison test was applied. The number of tested cells is shown in the bars. *, P < 0.05. (C) Effect of CaMex on PM targeting and activity of Ca2+ channels in COS1 cells expressing EYFPN-α1C, α2δ. (a) Whole-cell EYFP fluorescence. (Scale bar, 4 μm.) (b) Representative traces of _I_Ca recorded in response to the indicated 600-ms test pulses (_V_h = −90 mV). (D) Average I–V relationship for _I_Ca through the α1C/α2δ/CaMex channel (filled circles) coplotted with the voltage dependence of τ (open circles). _V_0.5 = 15.8 ± 0.8, _k_I–V = −9.1 ± 0.5, _E_rev = 110.3 ± 2.2 mV (n = 5). (E) Voltage dependence of activation of EYFPN-α1C/α2δ coexpressed with β2d (filled circles, n = 7) or CaMex (open circles, n = 9). Ca2+ tail currents (_I_tail) were recorded after repolarization for 10 ms to −50 mV following _V_t from −40 to +90 mV applied from _V_h = −90 mV for 20 ms. _I_tail were normalized to the peak _I_tail (_I_tail,max) and fitted with a Boltzmann equation: _I_tail/_I_tail,max = (_A_1 − _A_2)/[ 1 + exp(V − _V_a,50)/_k_a] + _A_2, were _V_a,50 is the half-maximal voltage for current activation, _k_a is the slope factor, _A_1 and _A_2 represent proportion of fully activated and nonactivated current. (F) Representative trace of _I_Ca through the CaMex-activated β-deficient Cav1.2 channel evoked by _V_t to +40 mV applied from _V_h = −90 mV for 30 s. (G) Averaged steady-state inactivation curve for _I_Ca through the EYFPN-α1C/α2δ/CaM channel (n = 5). One-second conditioning prepulses were applied from _V_h = −90 mV (up to +50 mV, 10-mV increments) followed by a 100-ms _V_t to +40 mV. The intervals between each cycle were 15 s. The peak current amplitudes in each curve were normalized to the maximum value determined in the range of −40 to +50 mV. The curves were fitted (smooth line) by Boltzmann function: I = A + B/(1 + exp[(V − _V_0.5,in)/_k_]), where A (0.50 ± 0.01) and B are fractions of noninactivating and inactivating currents, respectively, V is the conditioning prepulse voltage, _V_0.5,in = 10.3 ± 0.6 mV is the voltage at half-maximum of inactivation, and k = 5.4 ± 0.5 is a slope factor.

Fig. 3.

Fig. 3.

Effect of the replacement of Ca2+ for Ba2+ as the charge carrier through the EYFPN-α1C/α2δ channel modulated by CaMex. The EYFPN-α1C and α2δ subunits were coexpressed in COS1 cells with ECFPN-CaM. (A) Representative trace of the maximum _I_Ba evoked by _V_t = +30-mV applied for 600 ms from _V_h = −90 mV. For comparison, gray line shows the decay portion of the I_Ca trace (+30 mV, see Fig. 2_C) scaled to the same amplitude. (B) The averaged normalized I–V curve: _V_0.5 = 11.4 ± 1.5, _k_I–V = −9.4 ± 0.7, _E_rev = 93.5 ± 3.7 mV (n = 18). (C) Averaged steady-state inactivation curve for _I_Ba: A = 0.67 ± 0.01, _V_0.5,in = 15.4 ± 1.6 mV; k = 6.9 ± 1.4 (n = 5).

Fig. 4.

Fig. 4.

Ca2+-insensitive CaM1234 mutant supports gating of the Cavβ-subunit-deficient Cav1.2 calcium channel. The EYFPN-α1C and α2δ subunits were coexpressed in COS1 cells with CaM1234. (A) Epifluorescent image of an expressing cell showing PM targeting of EYFPN-α1C (arrows). (Scale bar, 4 μm.) (B) The averaged I–V curve (filled circles) coplotted with voltage dependence of τ for _I_Ca (open circles): _V_0.5 = 15.8 ± 1.0, _k_I–V = −9.1 ± 0.6, _E_rev = 120.8 ± 3.5 mV (n = 7). (C) The averaged steady-state inactivation curve for _I_Ca: A = 0.52 ± 0.01, _V_0.5,in = 14.5 ± 0.4 mV; k = 8.8 ± 0.4 (n = 6). (D) Averaged normalized voltage dependence of activation of _I_Ca through α1C/α2δ coexpressed with β2d (filled circles; n = 4) or CaM1234 (open circles; n = 4). (E) Representative trace of the maximum _I_Ca activated by _V_t to + 40 mV applied for 600 ms from _V_h = −90 mV. For comparison, the gray line shows a decay portion of _I_Ca through the β-deficient α1C/α2δ/CaMex channel evoked by V_t = +40-mV (for original trace, see Fig. 2_C).

Fig. 5.

Fig. 5.

Competition between CaMex and Cavβ for interaction with α1C/α2δ. (A) Western blot analysis (representing two independent experiments) of coimmunoprecipitation (IP) of CaMex with α1C in the absence (lane 1) or presence (2-4) of β2d. FLAGN-α1C and α2δ were coexpressed in COS1 cells with Venus (ct, control), ECFPN-CaM (1), Venus-β2d (2), or ECFPN-CaM + Venus-β2d (3, 4) (Right). Cells were lysed in the presence of 10 μM (ct, 1-3) or zero Ca2+ (4) and coIP with anti-FLAG Ab (Left). The expressed proteins (see Left) were analyzed with anti-FLAG (Upper) or anti-LC Ab (Lower). Molecular mass standards (in kDa) are indicated on the right. (B–E) Inhibition of CaMex modulation of the Cavβ-free Ca2+ channels by mutation of major CDI- or Cavβ-related α1C functional motifs. (B) α1C,L (LA-), (C) α1C,K (IQ-), (D) α1C,ΔLK (LA+IQ-deficient), or (E) mVenusN-α1CAIDM α1C subunits were coexpressed with α2δ and either ECFPN-CaM or β2d (a) or (b). Shown are representative traces (n = 3–10) of maximal _I_Ca recorded in response to _V_t = +30 (C and D) or +20 mV (B and E). V_h = −90 mV. (c) Distribution of EYFPN-α1CAIDM between PM and the cytoplasm in the presence of CaMex or β2d as compared with that for EYFPN-α1C in the presence of β2d (see Fig. 2_B). Number of tested cells is shown in the bars. *, P < 0.05.

Fig. 6.

Fig. 6.

CaMex does not induce endogenous Cavβ subunits in COS1 cells. (A) Lack of effect of CaMex on relative mRNA levels of endogenous Cavβ in COS1 cells. Each image represents a real-time PCR assessment (mean ± SEM, n = 5) of the mRNA levels (relative to GAPDH mRNA) of three major Cavβ subunits in nontransfected COS1 cells (NT) or those coexpressing α1C and α2δ with the EYFP mutant Venus (−CaM) or ECFPN-CaM (+CaM) under standard conditions used for electrophysiological experiments (Methods). PCR primers were designed to invariant exons of the monkey β1, β2, and β3 subunit genes. *, P < 0.05; **, _P_ > 0.05. (B) Lack of effect of CaMex on endogenous Cavβ binding to α1C revealed by coimmunoprecipitation analysis. FLAGN-α1C and α2δ were coexpressed in ≈106 COS1 cells with (lane 1) Venus, (lane 2) ECFPN-CaM, or (lane 3) human β1b (GenBank accession no. M92302, a), β2d (GenBank accession no. AF423191, b), or β3 subunit (GenBank accession no. X76555, c). α1C was identified on Western blot by anti-FLAG Ab (Upper). Cavβ subunits were identified (Lower) by Abs generated against rat/rabbit/human epitopes common with monkey: β1 (GenBank accession no. XM_001085813, monkey amino acids 19–34, a), β2 (GenBank accession no. XM_001092601, 387–410, b), and β3 (GenBank accession no. XM_001102938, 477–491, c). Molecular mass calibration in kDa is shown at right.

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