A Deletion Mutation in Na_v1.5 Impairs Calmodulin Interactions and Current Properties in a Compound Heterozygote Patient with Severe Conduction Disease

Background:
The role of Calmodulin (CaM) and the auxiliary Na_Vβ in the regulation of Na_V1.5 is not fully understood. A proband with severe cardiac conduction disorder, exhibited compound heterozygosity mutations found in two putative CaM SCN5A binding domains, K1493del in DIII-IV linker and A1924T in the C-terminus of Na_V1.5.

Methods:
Properties of WT and mutated Na_V1.5 currents were studied in HEK293 cells using whole-cell patch clamp technique. In order to assess the channel protein level, cell surface and total expression were quantified using biotinylation and western blot analysis. Na_V1.5 interactions with CaM were tested in a pull-down assay using CaM -sepharose beads.

Results:
Expression of Na_V1.5-K1493del, without an auxiliary Na_Vβ subunit, led to channel’s loss-of function (-9.3 ± 1.5 pA/pF n=7 vs. WT -284.8 ± 32.5 pA/pF n=21; p<0.001). In addition, a reduction in total channel expression to 33 ± 8% (n=10) compared to Na_V1.5-WT protein levels, was observed, with no change in the trafficking. Co-expression of the Na_Vβ1 subunit increased K1493del I_Na (-283.4 ± 31.1 pA/pF n=22) and total protein levels. In the presence of Ca²⁺, CaM interaction with K1493del was 4-5-fold smaller than with Na_V1.5-WT and A1924T. In low Ca²⁺ conditions, a leftward shift in activation curve was observed in K1493del (V_1/2 -41.9 ± 1.2 mV n=16 vs. WT -36.2 ± 1.3 mV n=8; p=0.008) . Steady-state inactivation curve show a typical leftward shift in low vs. high Ca²⁺ conditions for both WT and K1493del.

Conclusion:
Current and protein levels of Nav1.5 K1943 del were rescued by co-expression with Na_Vβ1. K1493del impairs channel biogenesis and CaM interaction.Suggesting a complex interaction domain including CaM, C-terminal and DIII-IV linker. These findings provide new insight in our understanding of the role of CaM and Na_Vβ in Na_V1.5 physiology in health and disease such as in our patient.