Accelerated inactivation of the L-type calcium current due to a mutation in CACNB2b underlies Brugada syndrome

Abstract

Recent studies have demonstrated an association between mutations in CACNA1c or CACNB2b and Brugada syndrome (BrS). Previously described mutations all caused a loss of function secondary to a reduction of peak calcium current (I(Ca)). We describe a novel CACNB2b mutation associated with BrS in which loss of function is caused by accelerated inactivation of I(Ca). The proband, a 32 year old male, displayed a Type I ST segment elevation in two right precordial ECG leads following a procainamide challenge. EP study was positive with induction of polymorphic VT/VF. Interrogation of implanted ICD revealed brief episodes of very rapid ventricular tachycardia. He was also diagnosed with vasovagal syncope. Genomic DNA was isolated from lymphocytes. All exons and intron borders of 15 ion channel genes were amplified and sequenced. The only mutation uncovered was a missense mutation (T11I) in CACNB2b. We expressed WT or T11I CACNB2b in TSA201 cells co-transfected with WT CACNA1c and CACNA2d. Patch clamp analysis showed no significant difference between WT and T11I in peak I(Ca) density, steady-state inactivation or recovery from inactivation. However, both fast and slow decays of I(Ca) were significantly faster in mutant channels between 0 and + 20 mV. Action potential voltage clamp experiments showed that total charge was reduced by almost half compared to WT. We report the first BrS mutation in CaCNB2b resulting in accelerated inactivation of L-type calcium channel current. Our results suggest that the faster current decay results in a loss-of-function responsible for the Brugada phenotype

Figure 1

Electrocardiograms of the patient. (A) Twelve-lead ECG of the patient at rest. ST-segment elevation and negative T-wave are present in one right precordial lead (V1). (B) After infusion of procainamide, a type ST segment elevation was apparent in V2 as well.. (C) Development of polymorphic ventricular tachycardia following programmed electrical stimulation (double extrastimuli).

Figure 1

Electrocardiograms of the patient. (A) Twelve-lead ECG of the patient at rest. ST-segment elevation and negative T-wave are present in one right precordial lead (V1). (B) After infusion of procainamide, a type ST segment elevation was apparent in V2 as well.. (C) Development of polymorphic ventricular tachycardia following programmed electrical stimulation (double extrastimuli).

Figure 2

A: DNA sequencing analysis. C to T substitution in exon 1 of CACNB2b predicts an amino acid substitution of threonine for lysine at codon 11 (T11I). B: Location of the T11I in the $-subunit of Ca_v 1.2. The cardiac Ca²⁺ channel α-subunit consists of four domains each containing six transmembrane-spanning segments.

Figure 3

Representative whole cell current recordings from a WT (A) and T11I mutant (B) expressed in TSA201 cells. Current recordings were obtained at test potentials between −50 and +60 mV in 10 mV increments from a holding potential of −90 mV. C: I–V relation for WT (n=12) and T11I (n=13) cells showing no statistically significant differences in peak calcium channel current density. D: Steady state-activation relation for WT and T11I. Chord conductance was determined using the ratio of current to the electromotive potential for the cells shown in Panel C. Data were normalized and plotted against their test potential.

Figure 4

Representative steady-state inactivation recordings for WT (A) and T11I (B) observed in response to the voltage clamp protocol shown at the top of the figure. C: Steady state-inactivation relation. Peak currents were normalized to their respective maximum values and plotted against the conditioning potential. T11I channels showed a mid-inactivation potential that was slightly but significantly hyperpolarized compared to WT channels.

Figure 5

Representative traces recorded from a WT (A) and T11I mutant (B) showing recovery of I_Ca. Recovery was measured using two identical voltage clamp steps to +20 mV from a holding potential of −90 mV separated by selected time intervals. Recovery time-course fit to a single exponential showed no difference in recovery rate between WT and T11I channels (C).

Figure 6

A. Inactivation time constants (τ) for the fast phase of I_Ca decay as a function of voltage. Inactivation time constants (τ) values were measured by fitting a biexponential function to the current decay. *p<0.05 vs WT. B: Inactivation time constants (τ) for the slow phase of I_Ca decay as a function of voltage. *p<0.05 vs WT.

Figure 7

Representative I_Ca currents elicited during action potential clamp experiments in TSA201 cells co-transfected with either WT or T11I CACNB2b. The action potentials were recorded from canine epicardial and endocardial cells which then served as the action potential clamp waveform (shown at top of figure). Horizontal line represents 0 mV. A: WT current traces following application of an epicardial and endocardial waveform. B: T11I currents following application of an epicardial and endocardial waveform. C: Superimposed current traces recorded from WT and T11I channels. Total charge during the plateau of the action potential was reduced by 42±2% (n=5) in T11I channels compared to WT when the epicardial waveform was applied. D: Superimposed current traces recorded from WT and T11I channels in 2 mM Ca²⁺ external and 36° C. Total charge during the plateau of the action potential was reduced by 51±6.7% (n=4, p<0.05) in T11I channels compared to WT when the epicardial waveform was applied.