Signalling events downstream of Gα_i stimulation.

<p>Representative western blot of cardiac tissues of animals treated either with saline or CCh (0.5 mg/kg body weight) for 15 min. (A) Western Blot and densitometry analyses of ventricular homogenates show that Akt phosphorylation was significantly and to a similar extent increased after CCh stimulation in all genotypes (n = 3). Total amount of Akt protein was unaltered and was used as loading control. (B) Western blot showing ERK1/2 phosphorylation and total ERK1/2 expression and bar graphs of combined results expressed as increase in pERK1/2 normalized to total ERK1/2 and compared to saline treated WT mice. The increase of ERK1/2 activation in CCh treated cells was markedly inhibited by Gα_i2 gene deletion compared to WT and Gα_i3^−/− mice (n = 3). *p<0.05 vs. WT NaCl.</p

Single-channel gating in WT and Gα_i3^−/− myocytes.

<p>Analysis of single-channel gating parameters in ventricular myocytes from WT (n = 7) and Gα_i3^−/− (n = 6) mice. Recordings with more than one channel were excluded from the analysis. i: unitary current; I_peak: peak ensemble average current; f_active: fraction of traces showing at least one opening; P_open: open probability in active traces; t_open: mean duration of openings; τ_open: dwell time constant of the open state; t_closed: mean duration between two consecutive openings; τ_closed,1: dwell time constant of the fast closed time component; τ_closed,2: dwell time constant of the slow closed time component; proportion: ratio of events conferring to either the fast or the slow closed time component; fl: mean latency until the first opening.</p><p>*: p<0.05 vs. WT.</p

Single-channel properties of L-type calcium channels.

<p>(A) Exemplary traces of barium currents show increased single-channel activity in ventricular myocytes of Gα_i3^−/− animals vs. WT mice. (B) The peak ensemble average current is −61±13 fA in Gα_i3^−/− (n = 6) and −44±9 fA in WT mice (n = 7). (C) The open probability within active sweeps is slightly enhanced in Gα_i3^−/− (5.4±1.0% vs. 4.0±0.9% WT) whereas (D) the mean closed time is significantly reduced (Gα_i3^−/− 3.8±0.5 ms vs. 6.6±0.9 ms WT). Unitary amplitude was not different with −0.83±0.02 pA (WT) and −0.79±0.03 pA (Gα_i3^−/−). *p<0.05 vs. WT. Box-and-whisker plots indicate minimum and maximum values as well as 25^th, 50^th and 75^th percentiles.</p

Cardiac whole-cell L-type calcium currents.

<p>Representative original current traces obtained at different test potentials (A) and IV-curves (B) reveal an increase of calcium current in ventricular myocytes from mice lacking Gα_i3 and a decrease in mice lacking Gα_i2 as compared to WT mice. (C) Steady-state inactivation of Gα_i2^−/− (n = 11) is shifted to more negative voltages as compared to Gα_i3^−/− (n = 13, p<0.05) and WT (n = 18, p<0.05). (D) A slowing of the recovery time constant τ is found in cardiomyocytes from Gα_i2^−/− mice (287±21 ms, n = 9) as compared to Gα_i3^−/− cells (n = 9) and WT (n = 16).</p

Effects of acute inactivation of G_i/o proteins by PTX incubation of isolated cardiac myocytes.

<p>(A) Effects of PTX on peak L-VDCC current density. PTX treatment by itself did not affect calcium current density. (B) Effects of PTX on steady-state inactivation, as gauged by the midpoint voltage V_0.5 of a Boltzmann function. No change is seen after 3 hours of drug-free incubation compared to 0 hour. PTX leads to a significant leftward shift of V_0.5 in WT (from −19.2±0.7 mV to −21.0±0.7 mV, n = 7–18) and Gα_i3^−/− (from −18.2±0.7 mV to −22.7±1.1 mV, n = 11–13). (C) PTX affects the recovery of the L-VDCC from inactivation. PTX inhibits the channel recovery in Gα_i3^−/− (τ from 189±12 ms to 350±26 ms, n = 5–11). *p<0.05 vs. WT, ^†p<0.05 vs. Gα_i3^−/−, ^¶p<0.05 vs. 3 h without PTX.</p

Steady-state inactivation parameters.

<p>In Gα_i2^−/− cardiomyocytes V_0.5 of the steady-state inactivation is significantly shifted to more negative potentials. In addition, Boltzmann fits revealed a significant flattening of the curves as indicated by increased slope factors.</p><p>*p<0.05 vs. WT,</p>†<p>p<0.05 vs. Gα_i3^−/−.</p