Constitutively Activating GNAS Somatic Mutation in Right Ventricular Outflow Tract Tachycardia

Abstract

[Figure: see text].

Keywords: GTP-binding proteins; arrhythmia; mutation; signal transduction; tachycardia, ventricular.

Figure 1.

Sequence of polymerase chain reaction-amplified DNA from right ventricular apex (RVA) and right ventricular outflow tract (RVOT) biopsy samples. A, Novel point mutation (W234R) in the switch II region of G_sα was identified from a biopsy sample obtained from the site of origin of ventricular tachycardia in the RVOT, but not from the RVA. B, G_sα protein sequence alignment showing site of point mutation (W234R) within a conserved region across several species.

Figure 1.

Sequence of polymerase chain reaction-amplified DNA from right ventricular apex (RVA) and right ventricular outflow tract (RVOT) biopsy samples. A, Novel point mutation (W234R) in the switch II region of G_sα was identified from a biopsy sample obtained from the site of origin of ventricular tachycardia in the RVOT, but not from the RVA. B, G_sα protein sequence alignment showing site of point mutation (W234R) within a conserved region across several species.

Figure 2.

Effects of W234R mutation in G_sα (stimulatory G protein alpha- subunit) on cyclic AMP (cAMP) cellular content. S49 cycˉ G_sα deficient cells were transfected with either wild-type or W234R mutant G_sα cDNA. Western blot demonstrated similar expression levels of both wild-type and mutant protein. cAMP levels were assayed before and after treatment with isoproterenol (ISO). Three separate experiments for each construct were carried out in triplicate. Thus, there are 9 data points for each condition. Basal cAMP levels in cells transfected with wild-type G_sα were 5.0±0.7 pmol/10⁷ cells, whereas basal cAMP levels in cells transfected with mutant G_sα were 79±12 pmol/10⁷ cells, P<0.001. * P< 0.001 for comparison of basal cAMP levels among the three constructs. ** P<0.001 for comparison of cAMP levels after treatment with ISO across all three constructs.

Figure 3.

Structural and functional properties of wild-type and mutant W234R G_sα recombinant proteins. A, Wild-type G_sα is protected from trypsin proteolysis after incubation with GTPγS (43kDa proteolytic fragments accumulate) but not with GTP. B, Left panel. Recombinant wild-type and mutant G_sα proteins were incubated with ³⁵[S]GTPγS and analyzed as described in Methods. No effect of mutation on GTP binding was observed and similar K_d values were calculated for both proteins (P=0.46). B, Right panel. Wild-type and mutant G_sα proteins were incubated with [γ−³²P]GTP. Aliquots (50 μl) were analyzed for [³²P]Pi at the indicted time. k_cat was calculated by observation of a single round of GTP hydrolysis performed in triplicate experiments.

Figure 3.

Structural and functional properties of wild-type and mutant W234R G_sα recombinant proteins. A, Wild-type G_sα is protected from trypsin proteolysis after incubation with GTPγS (43kDa proteolytic fragments accumulate) but not with GTP. B, Left panel. Recombinant wild-type and mutant G_sα proteins were incubated with ³⁵[S]GTPγS and analyzed as described in Methods. No effect of mutation on GTP binding was observed and similar K_d values were calculated for both proteins (P=0.46). B, Right panel. Wild-type and mutant G_sα proteins were incubated with [γ−³²P]GTP. Aliquots (50 μl) were analyzed for [³²P]Pi at the indicted time. k_cat was calculated by observation of a single round of GTP hydrolysis performed in triplicate experiments.

Figure 4.

Ribbon diagram for the Ras-like domain of bovine Gsα (PDB ID 1AZT), with W234 mutated to R (red sticks, label underlined). The bound GTP analog is colored according to atom types, and surrounding secondary structure elements are labeled according to Sunahara et al. Catalytically important residues are shown in yellow, and the switch II helix α2 in cyan.

Figure 5.

Whole-cell patch clamp. A, Exemplar traces measured using whole-cell patch clamp from CHO cells expressing cardiac L-type calcium channel subunits (α1, β2a and α2-δ) and wild-type or W234R human G_sα as indicated. Zero current level indicated by dashed line. Voltage protocol shown in lower inset. Arrow, approximate time point at which peak current was measured. B. Mean current density-voltage relationships for cells as in panel A; n = 27 (wild-type) and n = 24 (W234R). *P < 0.05 comparing wild-type versus W234R currents.

Figure 6.

Model prediction of proarrhythmic electrical activity due to increased L-type calcium current secondary to W234R G_sα mutation. Simulations of wild-type ionic behavior in response to 2 Hz pacing for 30 s followed by a pacing pause results in normal action potentials (black trace). Simulating the W234R G_sα mutation causes action potential prolongation (50% increase in the permeability of the L-type calcium current: blue trace). Challenging the cell by increasing the calcium current further leads to multiple DADs (arrows) and a spontaneously triggered action potential (100% increase in the permeability of the L-type calcium current; red trace).

Figure 7.

Schematic showing the signal transduction cascade that mediates ventricular tachycardia due to cAMP-mediated triggered activity. Clinical right ventricular outflow tract tachycardia is usually adrenergically-mediated, and is frequently triggered by exercise or stress. In contrast, the somatic mutation W234R G_sα doesn’t require an agonist for activation since it is constitutively active, accounting for resting ventricular tachycardia. Abbreviations: AC, adenylyl cyclase; A1AR, adenosine receptor A1; ACH; acetylcholine; ADO, adenosine; β-AR, β-adrenergic receptor; CICR, calcium-induced calcium release; DAD, delayed afterdepolarization; G_i2α, inhibitory G-protein; G_sα, stimulatory G-protein; I_ti, transient inward current; LTCC, L-type calcium channel; mAChR, muscarinic acetylcholine receptor; NCX, sodium–calcium exchanger; PKA, cAMP-dependent protein kinase (protein kinase A); PLB, phospholamban; RyR2, ryanodine receptor; SERCA, sarco/endoplamic reticulum Ca²⁺-ATPase; SR, sarcoplasmic reticulum.