Four and a half LIM protein 1: A partner for KCNA5 in human atrium

AIMS: Protein-protein interactions are critical for the normal membrane trafficking, localization, and function of voltage-gated ion channels. In human heart, the Shaker-related voltage-gated K(+) channel KCNA5 alpha-subunit forms the major basis for an atrial-specific, ultra-rapid delayed rectifier K(+) current, I(Kur). We sought to identify proteins that interact with KCNA5 in human atrium and to investigate their role in the I(Kur) complex. Methods and Results Using a glutathione-S-transferase (GST)-KCNA5 C-terminal fusion protein and mass spectrometry-based methods, the scaffolding protein four and a half LIM (for Lin-11, Isl-1, and Mec3) protein 1 (FHL1) was identified as a potential protein partner for KCNA5. Immunoprecipitation experiments confirmed a physical interaction of FHL1 with the K(+) channel complex in human atrium, as well as in Chinese hamster ovary (CHO) cells transfected with both KCNA5 and FHL1. In cotransfected cells, confocal microscopy demonstrated areas of colocalization after immunolabeling both proteins. To investigate the functional effects of this interaction, K(+) currents were recorded in CHO cells transfected with KCNA5 in the absence and presence of FHL1 coexpression. With coexpression of FHL1, K(+) current density was markedly increased, compared to cells expressing KCNA5 alone. This effect was associated with a shift in the voltage dependence of K(+) channel activation to more positive potentials, consistent with findings for I(Kur) in atrial myocytes. FHL1 also increased the extent and speed of K(+) current slow inactivation, with additional effects on the voltage dependence and recovery of this process. CONCLUSIONS: These results support a role for FHL1 as a key molecular component in the I(Kur) complex in human atrium, where it likely regulates functional expression of KCNA5.