Effects of KCL on Calmodulin Mutants Defective in Ion Channel Regulation

Abstract

Calmodulin (CaM) is an essential eukaryotic calcium sensor that regulates many ion channels and enzymes. CaM is comprised of two homologous domains (N and C), each with two calcium-binding sites. Paramecium mutants identified by a genetic screen to be defective in response to external stimuli showed that the two domains of CaM have different effects on ion channel regulation. Under-reactive mutants (changes in the N-domain of CaM) affect regulation of a calcium-dependent Na+ current, while over-reactive mutants (changes within the C-domain) affect a calcium-induced K+ current. Because CaM binds to the intracellular regions of these channels, it is subject to changing concentrations of Na+ and K+. This study explores the effects of potassium on the domain-specific conformation and calcium-binding energetics of under- and over-reactive mutants. Potassium-induced changes in altered thermal stability of apo (calcium-depleted) CaM explored effects on tertiary structure. Fluorescence-monitored calcium titrations over the range of 0 to 300 mM KCl showed that the total free energy of binding calcium to each domain became less favorable by about 2.5 kcal/mol. In thermal denaturation studies of apo PCaM, the melting temperature (Tm) increased by approximately 5°C and the enthalpy (ΔH) changed by 3.5 kcal/mol when [KCl] increased from 50 to 300 mM. The findings indicate that potassium ions increased tertiary constraints on apo CaM, making it less flexible. Linkage relationships resulted in lowering calcium-binding affinity. Thus, an influx of K+ through an ion channel would shift the equilibrium of CaM towards the apo state. This effect would be exacerbated for over-reactive mutants that have intrinsically lower calcium affinity than wild-type CaM.