Addition, mutations in genes otherthan SCN5A have been identified in a low proportion of BrS patients [8]. The Nav1.5 protein, with 2016 amino acids and a molecular weight of 227 kDa, consists of four homologous domains (DI-DIV) [9]. Each domain contains six transmembrane Genz 99067 web segments (S1 6) linked by intracellular and extracellular loops. S4 segments contain 5 positively charged residues (arginine or lysine) separated by 2 hydrophobic residues, and form the voltage MedChemExpress EAI045 sensor domain of the channel. The pore region of the channel is formed by the interaction among segments S5, S6 and loop S5 6 of domains DI to DIV [10]. The pore (P)-helices that stabilize the Na+ ion in the central cavity are formed by the loops S5 6 [11]. In the present study, we aimed to characterize the biophysical properties of Nav1.5 channels carrying a novel mutation, I890T, in the first P-helix of DII to establish whether this mutation is associated with BrS. We show evidence of loss-of-function of the mutant Nav1.5 channel, which 18325633 is consistent with the patient’s clinical manifestation of BrS.Novel Nav1.5 Pore Mutation I890T Causes BrSMethods Ethics StatementThis study was approved by the Ethics Committee of Hospital Josep Trueta (Girona, Spain) and conforms with the principles outlined in the Declaration of Helsinki. All individuals signed a written informed consent to participate in the study.Electrophysiological StudiesSodium currents were measured at room temperature using the standard whole cell patch-clamp technique [17]. Voltage clamp experiments were controlled and analyzed with an Axopatch 200B amplifier and pClamp 10.2/Digidata 1440A acquisition system (Molecular Devices, Sunnyvale, CA, USA) and OriginPro8 software (OriginLab Corporation, Northampton, MA, USA). The bath solution contained (mM): 140 NaCl, 3 KCl, 10 N-2hydroxyethylpiperazine- N’ -2-ethanesulfonic acid (HEPES), 1.8 CaCl2 and 1.2 MgCl2 (pH 7.4, NaOH); and the pipette solution (mM): 130 CsCl, 1 Ethylene glycol-bis(2-amino-ethylether)-N,N, N’,N’-tetra-acetic acid (EGTA), 10 HEPES, 10 NaCl and 2 ATP Mg2+ (pH 7.2, CsOH). Osmolality was adjusted by the addition of glucose to 326 and 308 mOsm for bath and pipette solution, respectively. Pipettes were pulled from glass capillaries (Brand GMBH+CO KG, Wertheim, Germany) and their resistance ranged from 2.5 to 3.2 MV when filled with the internal solution. 80?0 series resistance compensation was used during whole cell measurements. Membrane potentials were not corrected for junction potentials that arose between the pipette and bath solution. Data were filtered at 5 kHz and sampled at 5?0 kHz. Activation curve data were fitted to a Boltzmann equation, of the form g = gmax/(1+ exp(V1/22Vm)/k), where g is the conductance, gmax the maximum conductance, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are activated and k is the slope factor. Steady-state inactivation values were fitted to a Boltzmann equation of the form I = Imax/(1+ exp(V1/22Vm)/k), where I is the peak current amplitude, Imax the maximum peak current amplitude, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are inactivated, and k is the slope factor. The sodium current decay after the peak INa was fitted with a monoexponential function between 240 and 225 mV, and a bi-exponential function between 220 and 20 mV, from where t fast and t slow were obtained. Both the slow inactivation and the recovery from inactivation data were fitt.Addition, mutations in genes otherthan SCN5A have been identified in a low proportion of BrS patients [8]. The Nav1.5 protein, with 2016 amino acids and a molecular weight of 227 kDa, consists of four homologous domains (DI-DIV) [9]. Each domain contains six transmembrane segments (S1 6) linked by intracellular and extracellular loops. S4 segments contain 5 positively charged residues (arginine or lysine) separated by 2 hydrophobic residues, and form the voltage sensor domain of the channel. The pore region of the channel is formed by the interaction among segments S5, S6 and loop S5 6 of domains DI to DIV [10]. The pore (P)-helices that stabilize the Na+ ion in the central cavity are formed by the loops S5 6 [11]. In the present study, we aimed to characterize the biophysical properties of Nav1.5 channels carrying a novel mutation, I890T, in the first P-helix of DII to establish whether this mutation is associated with BrS. We show evidence of loss-of-function of the mutant Nav1.5 channel, which 18325633 is consistent with the patient’s clinical manifestation of BrS.Novel Nav1.5 Pore Mutation I890T Causes BrSMethods Ethics StatementThis study was approved by the Ethics Committee of Hospital Josep Trueta (Girona, Spain) and conforms with the principles outlined in the Declaration of Helsinki. All individuals signed a written informed consent to participate in the study.Electrophysiological StudiesSodium currents were measured at room temperature using the standard whole cell patch-clamp technique [17]. Voltage clamp experiments were controlled and analyzed with an Axopatch 200B amplifier and pClamp 10.2/Digidata 1440A acquisition system (Molecular Devices, Sunnyvale, CA, USA) and OriginPro8 software (OriginLab Corporation, Northampton, MA, USA). The bath solution contained (mM): 140 NaCl, 3 KCl, 10 N-2hydroxyethylpiperazine- N’ -2-ethanesulfonic acid (HEPES), 1.8 CaCl2 and 1.2 MgCl2 (pH 7.4, NaOH); and the pipette solution (mM): 130 CsCl, 1 Ethylene glycol-bis(2-amino-ethylether)-N,N, N’,N’-tetra-acetic acid (EGTA), 10 HEPES, 10 NaCl and 2 ATP Mg2+ (pH 7.2, CsOH). Osmolality was adjusted by the addition of glucose to 326 and 308 mOsm for bath and pipette solution, respectively. Pipettes were pulled from glass capillaries (Brand GMBH+CO KG, Wertheim, Germany) and their resistance ranged from 2.5 to 3.2 MV when filled with the internal solution. 80?0 series resistance compensation was used during whole cell measurements. Membrane potentials were not corrected for junction potentials that arose between the pipette and bath solution. Data were filtered at 5 kHz and sampled at 5?0 kHz. Activation curve data were fitted to a Boltzmann equation, of the form g = gmax/(1+ exp(V1/22Vm)/k), where g is the conductance, gmax the maximum conductance, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are activated and k is the slope factor. Steady-state inactivation values were fitted to a Boltzmann equation of the form I = Imax/(1+ exp(V1/22Vm)/k), where I is the peak current amplitude, Imax the maximum peak current amplitude, Vm is the membrane potential, V1/2 is the voltage at which half of the channels are inactivated, and k is the slope factor. The sodium current decay after the peak INa was fitted with a monoexponential function between 240 and 225 mV, and a bi-exponential function between 220 and 20 mV, from where t fast and t slow were obtained. Both the slow inactivation and the recovery from inactivation data were fitt.
