Background:
Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca
2+
-dependent ion channels. Mutations in CALM1, CALM2, and CALM3 have recently been linked to cardiac arrhythmias, such as Long QT Syndrome (LQTS), catecholaminergic polymorphic ventricular tachycardia (CPVT), and familial idiopathic ventricular fibrillation (IVF). Small-conductance Ca
2+
- activated K
+
channels (SK) are voltage-independent channels that are regulated solely from beat-to-beat changes in intracellular calcium. CaM regulates the function of multiple ion channels, including SK channels, although the effect of CaM mutations on these channels is not yet understood. We hypothesize that human CaM mutations linked to sudden cardiac death disrupt SK channel function by distinct mechanisms.
Methods and Results:
We tested the effects of LQTS (CaM
D96V
, CaM
D130G
), CPVT (CaM
N54I
, CaM
N98S
), and IVF (CaM
F90L
) CaM mutants compared to CaM
WT
on SK channel function. Using whole-cell voltage-clamp recordings, we found that CaM
D96V
and CaM
D130G
mutants significantly inhibited apamin-sensitive currents. Similarly, action potential studies in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) also revealed significant knockdown of apamin-sensitive currents.
Immunofluorescent confocal microscopy confirmed that this effect was not due to changes in SK channel trafficking. Rather, co-immunoprecipitation studies showed a significant decrease in the association of these CaM mutants with the SK channel. Rosetta molecular modeling was used to identify a conformational change in CaM
F90L
structure compared to that of CaM
WT
.
Conclusions:
We found that CaM
D96V
and CaM
D130G
mutants significantly reduced apamin-sensitive currents, through a dominant negative effect on SK channel function. Consistent with our hypothesis, CaM
F90L
resulted in the least inhibitory effects. The data suggests that specific mutations with phenylalanine to leucine (CaM
F90L
) may disrupt the interaction between apo-CaM with CaMBD on the SK2 channel.
1P363 An attempt to visualize conformational changes during channel gating in the bacterial mechanosensitive channel, MscL(14. Ion channels and receptors,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)
