Human SCN5A Stable Cell Line - HEK293

In cardiomyocytes, the voltage-gated transient outward potassium current (Ito) is responsible for Phase 1 repolarization of the cardiac action potential (AP). Gain-of-function mutations in KCND3-the gene encoding the Kv4.3 channel that carries Ito-have been established as being associated with Brugada syndrome (BrS). Recent studies suggest that an augmentation of Ito may directly impact cardiac conduction function. Here, researchers investigated the effects of Kv4.3 overexpression on Nav1.5 currents and the consequent availability of sodium channels. The cardiac sodium current (INa), generated by the voltage-gated sodium channel Nav1.5 (encoded by the SCN5A gene), is responsible for driving the rapid upstroke phase of the cardiac action potential (AP). The researchers found that in HEK293 cells stably expressing Nav1.5 (i.e., HEK293-Nav1.5 cells), overexpression of the Kv4.3 protein significantly reduced Nav1.5 current density, yet did not alter its kinetic properties. Furthermore, measurements utilizing alternating voltage/current-clamp techniques revealed that Kv4.3 overexpression reduced the maximum upstroke velocity of action potentials in HEK293-Nav1.5 cells. These effects induced by Kv4.3 could not be explained solely by changes in the total expression levels of the Nav1.5 protein. Through further simulations utilizing multicellular computational models, the researchers confirmed that the experimentally observed phenomenon-namely, the simultaneous augmentation of Kv4.3 currents and attenuation of Nav1.5 currents-could potentially lead to cardiac conduction block, thereby underscoring the potential functional significance of the findings presented in this study.

Here, researchers characterized the effects of Kv4.3 overexpression on Nav1.5-based currents in HEK293-Nav1.5 cells. Figure 1A displays representative Nav1.5 currents activated by depolarizing voltage-clamp steps-starting from a holding potential of −120 mV, with 5 mV increments and a duration of 500 ms-in HEK293-Nav1.5 cells transfected with either IRES-GFP or KCND3-IRES-GFP. Under both conditions, Nav1.5 currents began to activate around −60 mV, reached a peak around −30 mV, and subsequently declined in amplitude due to the diminishing driving force for Na+. Compared to cells transfected with IRES-GFP, cells transfected with KCND3-IRES-GFP exhibited significantly lower Nav1.5 current densities (Figure 1B). Next, the researchers determined whether the reduction in Nav1.5 current density was accompanied by alterations in gating properties. No significant differences were observed in the t50% (half-decay time) between cells transfected with IRES-GFP and those transfected with KCND3-IRES-GFP. To determine the voltage dependence of the activation process in cells transfected with IRES-GFP versus KCND3-IRES-GFP, the I-V relationship curves shown in Figure 1B were first corrected for the Na+ driving force. Notably, the Nav1.5 current reversal potential calculated using the Nernst equation was approximately +17.58 mV, a value consistent with the sodium current recordings shown in Figure 1A. Subsequently, the current amplitudes were normalized (relative to their maximum amplitudes), and the resulting curves were fitted with a Boltzmann distribution function. Figure 1C illustrates the superposition of the voltage-dependence curves for the inactivation process. These inactivation curves were constructed by normalizing the current amplitudes to the maximum current value elicited during the −20 mV voltage-clamp step. Similarly, Figure 1D illustrates the overlap of the voltage-dependence curves for the activation process in cells transfected with IRES-GFP and KCND3-IRES-GFP. These data indicate that neither the voltage dependence of activation nor that of inactivation of the Nav1.5 current was affected by the expression of Kv4.3.

Figure 1. Kv4.3 overexpression reduces Nav1.5 currents. (Portero, Vincent, et al., 2018)