Calcium/calmodulin-dependent serine protein kinase CASK modulates the L-type calcium current.

AIM: The L-type voltage-gated calcium channel Cav1.2 mediates the calcium influx into cells upon membrane depolarization. The list of cardiopathies associated to Cav1.2 dysfunctions highlights the importance of this channel in cardiac physiology. Calcium/calmodulin-dependent serine protein kinase (CASK), expressed in cardiac cells, has been identified as a regulator of Cav2.2 channels in neurons, but no experiments have been performed to investigate its role in Cav1.2 regulation. METHODS AND RESULTS: Full length or the distal C-terminal truncated of the pore-forming Cav1.2 channel (Cav1.2α1c), both present in cardiac cells, were expressed in TsA-201 cells. In addition, a shRNA silencer, or scramble as negative control, of CASK was co-transfected in order to silence CASK endogenously expressed. Three days post-transfection, the barium current was increased only for the truncated form without alteration of the steady state activation and inactivation biophysical properties. The calcium current, however, was increased after CASK silencing with both types of Cav1.2α1c subunits suggesting that, in absence of calcium, the distal C-terminal counteracts the CASK effect. Biochemistry experiments did not reveals neither an alteration of Cav1.2 channel protein expression after CASK silencing nor an interaction between Cav1.2α1c subunits and CASK. Nevertheless, after CASK silencing, single calcium channel recordings have shown an increase of the voltage-gated calcium channel Cav1.2 open probability explaining the increase of the whole-cell current. CONCLUSION: This study suggests CASK as a novel regulator of Cav1.2 via a modulation of the voltage-gated calcium channel Cav1.2 open probability.

Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I.

BACKGROUND: Progressive cardiac conduction disease (PCCD) is one of the most common cardiac conduction disturbances. It has been causally related to rare mutations in several genes including SCN5A, SCN1B, TRPM4, LMNA and GJA5. METHODS AND RESULTS: In this study, by applying targeted next-generation sequencing (NGS) in 95 unrelated patients with PCCD, we have identified 13 rare variants in the TRPM4 gene, two of which are currently absent from public databases. This gene encodes a cardiac calcium-activated cationic channel which precise role and importance in cardiac conduction and disease is still debated. One novel variant, TRPM4-p.I376T, is carried by the proband of a large French 4-generation pedigree. Systematic familial screening showed that a total of 13 family members carry the mutation, including 10 out of the 11 tested affected individuals versus only 1 out of the 21 unaffected ones. Functional and biochemical analyses were performed using HEK293 cells, in whole-cell patch-clamp configuration and Western blotting. TRPM4-p.I376T results in an increased current density concomitant to an augmented TRPM4 channel expression at the cell surface. CONCLUSIONS: This study is the first extensive NGS-based screening of TRPM4 coding variants in patients with PCCD. It reports the third largest pedigree diagnosed with isolated Progressive Familial Heart Block type I and confirms that this subtype of PCCD is caused by mutation-induced gain-of-expression and function of the TRPM4 ion channel.