In vivo expression of G-protein beta1gamma2 dimer in adult mouse skeletal muscle alters L-type calcium current and excitation-contraction coupling

Abstract

A number of G-protein-coupled receptors are expressed in skeletal muscle but their roles in muscle physiology and downstream effector systems remain poorly investigated. Here we explored the functional importance of the G-protein betagamma (Gbetagamma) signalling pathway on voltage-controlled Ca(2+) homeostasis in single isolated adult skeletal muscle fibres. A GFP-tagged Gbeta(1)gamma(2) dimer was expressed in vivo in mice muscle fibres. The GFP fluorescence pattern was consistent with a Gbeta(1)gamma(2) dimer localization in the transverse-tubule membrane. Membrane current and indo-1 fluorescence measurements performed under voltage-clamp conditions reveal a drastic reduction of both L-type Ca(2+) current density and of peak amplitude of the voltage-activated Ca(2+) transient in Gbeta(1)gamma(2)-expressing fibres. These effects were not observed upon expression of Gbeta(2)gamma(2), Gbeta(3)gamma(2) or Gbeta(4)gamma(2). Our data suggest that the G-protein beta(1)gamma(2) dimer may play an important regulatory role in skeletal muscle excitation-contraction coupling.

Figure 1. Expression of the GFP-tagged Gβ₁γ₂ dimer in adult muscle fibres

A, confocal images of the GFP fluorescence from a fibre freshly isolated from a muscle transfected with the cDNAs coding for the GFP–Gβ₁ fusion protein along with the Gγ₂ subunit. B, di-8-ANNEPS staining. C, overlay of the two images. D, normalized mean fluorescence GFP and di-8-ANNEPS profile along the x axis of the box region superimposed on the image in C. GFP–Gβ₁ fusion protein yields subcellular localization consistent with the T-tubule membrane. TS, T-tubule spacing; SL, sarcomere length.

Figure 3. Expression of G-protein β₂γ₂, β₃γ₂ or β₄γ₂ dimers does not alter L-type calcium currents in skeletal muscle fibres

A, representative sets of Ca²⁺ current traces recorded from a Gβ₂γ₂ (top panel), Gβ₃γ₂ (middle panel) and Gβ₄γ₂ (bottom panel) -expressing fibres in response to 1 s depolarizing steps to values ranging between −40 mV and +80 mV from a holding potential of −80 mV. B, corresponding mean voltage dependence of the peak Ca²⁺ current density. Inset presents the mean half-maximal activation potential for Gβ₂γ₂, Gβ₃γ₂ and Gβ₄γ₂-expressing fibres. C, corresponding mean values for the voltage dependence of Ca²⁺ conductance in the three populations. Inset presents the mean maximal conductance for Gβ₂γ₂, Gβ₃γ₂ and Gβ₄γ₂-expressing fibres. The dashed line corresponds to the values from the control fibres. D, expression pattern of the GFP-tagged Gβ₂γ₂, Gβ₃γ₂ and Gβ₄γ₂ dimers in adult muscle fibres. Each panel shows a region of interest selected from a confocal image of the GFP fluorescence (green) of a fibre expressing the given Gβ subunit. The fibre expressing Gβ₂γ₂ was also stained with di-8-ANNEPS and the corresponding fluorescent image is shown in red. The graph underneath each set of images shows the normalized fluorescence profile of GFP (and di-8-ANNEPS) along the x axis of the boxed region superimposed on each image.

Figure 2. Expression of G-protein β₁γ₂ dimers reduces L-type calcium current density in skeletal muscle fibres

A, representative sets of Ca²⁺ current traces recorded from a control (left panel) and from a Gβ₁γ₂ dimer-expressing fibre (right panel) in response to 1 s depolarizing steps to values ranging between −50 mV and +80 mV from a holding potential of −80 mV. B, corresponding mean voltage dependence of the peak Ca²⁺ current density. Inset presents the mean half-maximal activation potential for control and Gβ₁γ₂-expressing fibres. C, corresponding mean values for the voltage dependence of Ca²⁺ conductance in the two populations. Inset presents the mean maximal conductance for control and Gβ₁γ₂-expressing fibres. The maximal conductance was reduced by 35% (P= 0.001) in the Gβ₁γ₂-expressing fibres.

Figure 4. Expression of G-protein β₁γ₂ dimers slows down L-type calcium current activation and inactivation kinetics

A, normalized Ca²⁺ current traces recorded from a control and from a Gβ₁γ₂ dimer-expressing fibre in response to 1 s depolarizing steps to +10 mV (left panel) and +30 mV (right panel) from a holding potential of −80 mV. The continuous superimposed bold lines correspond to the results from fitting a single exponential function to the inactivating phase of the current. B, mean values for the time to peak of the Ca²⁺ current for control and Gβ₁γ₂ dimer-expressing fibres. Values were significantly increased in the Gβ₁γ₂ dimer-expressing fibres for potential values from 0 mV to +40 mV. C, mean values for the time constant τ of Ca²⁺ current decay. Values were significantly increased in the Gβ₁γ₂ dimer-expressing fibres at potentials ranging from 0 mV to +30 mV. D, dependence of the time constant of Ca²⁺ current decay at +10 mV upon the peak current density, in control and Gβ₁γ₂-expressing fibres. Values for the time constant were grouped into 4 classes of peak current density.

Figure 5. A strong depolarizing pre-pulse allows partial recovery from the slowing of L-type calcium current kinetics induced by Gβ₁γ₂

A, representative Ca²⁺ current traces recorded from a control (left panel) and from a Gβ₁γ₂ dimer-expressing fibre (right panel) in response to a 1 s depolarizing step to +20 mV from a holding potential of −80 mV, before (P1) and after (P2) a strong depolarizing pre-pulse (PP) to +100 mV. B, enlarged view of the Ca²⁺ current traces shown in A after normalization to the same peak amplitude, with (P2) and without (P1) pre-pulse. C, mean normalized values of P2/P1 peak Ca²⁺ current amplitude as a function of the pre-pulse (PP) duration. D, mean values for the time to peak of the Ca²⁺ current recorded in P2 as a function of the pre-pulse duration. The inset shows the corresponding mean values after normalization to the time to peak of the current measured in absence of pre-pulse. E, mean values for the time constant of decay of the Ca²⁺ current recorded in P2 as a function of the pre-pulse duration. The inset shows the corresponding mean values after normalization to the time constant measured in absence of pre-pulse.

Figure 6. Expression of G-protein β₁γ₂ dimers strongly alters the voltage-activated Ca²⁺ transient in skeletal muscle fibres

A, representative indo-1 [Ca²⁺] traces from a control (left panel) and from a Gβ₁γ₂ dimer-expressing fibre (right panel) in response to depolarizing steps of increasing duration to +10 mV from a holding potential of −80 mV. The inset in each panel shows the corresponding mean (continuous traces) ±s.e.m. (grey shading) [Ca²⁺] traces. B–D, corresponding mean ±s.e.m. values of peak change in [Ca²⁺], time constant (τ) of [Ca²⁺] decay after the end of the pulse and final change in [Ca²⁺] level, respectively, measured from 10 control fibres and 6 Gβ₁γ₂ dimer-expressing fibres. The peak change in [Ca²⁺] was reduced by 43% (P= 0.0003) (20 ms depolarizing step) in the Gβ₁γ₂-expressing fibres. Values for the final change in [Ca²⁺] level were from the exponential fits. When not visible, errors bars are smaller than the mean dot.

Figure 7. Intramembrane charge movement in Gβ₁γ₂-expressing fibres

A, representative charge movement records from a control fibre and from a GFP-Gβ₁γ₂-positive fibre, measured in response to 50 ms depolarizing steps to the indicated values of membrane potential. B, mean voltage distribution of the ‘on’ charge in control fibres (n= 8) and in GFP-Gβ₁γ₂-positive fibre (n= 5). Superimposed continuous lines correspond to a two-state Boltzmann distribution calculated using the mean parameters obtained from fits to the individual sets of data in the two groups of fibres (see text for details).