KChIP2 Serves Multiple Functions in Cardiac Myocytes in Splice Isoform-Dependent Manner

Abstract

K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearing Ca sensors that serve diverse functions. KChIP2 is the major KChIP expressed in the heart, that can modulate key cardiac channels (Itof, Nav, CaV and IKur) by directly interacting with their pore-forming subunits (Kv4, Nav1.5, Cav1.2, and Kv1.5, respectively) on the cell surface. More recent data suggest that KChIP2 can traffic to nuclei and exert transcriptional activity. Nine KChIP2 splice variants have been identified, that share C-terminal EF-hand-motifs but diverge in N-termini. Based on N-terminal sequences, KChIP2 splice variants can be broadly divided as palmitoylation-capable and palmitoylation-incapable. Questions: (1) Does palmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2 splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c (palmitoylation-capable and -incapable) with mCherry, (2) Express KChIP2 isoforms in COS-7 cells alone or with GFP-Kv4.3, (3) Track KChIP2a/c distribution and relationship with Kv4.3 with confocal microscopy, (4) Quantify Itof channel function and subcellular distribution with patch clamp and subcellular fractionation/immunoblots, (5) Quantify native KChIP2 and Kv4 proteins and Itof channel function in ventricular myocytes from young and old spontaneously hypertensive rats (SY and SO, 4-6 and 22-24 months respectively; the latter suffers from severe hypertrophy/heart failure). Results: (1) When expressed alone, KChIP2a is strong in vesicles, Golgi and plasma membrane (PM), while KChIP2c is strong in nuclei and weak in PM. Pharmacological inhibition of palmitoylation directs KChIP2a from PM to nuclei, but does not alter nuclei localization of KChIP2c. (2) Kv4.3 coexpression leads to Kv4.3/KChIP2a overlap in Golgi, post-Golgi vesicles and PM. KChIP2c and Kv4.3 overlap on PM. While both KChIP2a and KChIP2c accelerate Kv4.3 recovery from inactivation, KChIP2a but not KChIP2c increases the current amplitude. (3) Relative to SY, Itof density is markedly reduced in epicardial myocytes (Epi VMs) of SO hearts. Kv4.2 and Kv4.3 proteins are modestly or not lowered in Epi VMs. While KChIP2a protein is markedly reduced, KChIP2c protein is significantly increased. Conclusions: Reversible palmitoylation confers dynamic KChIP2a localization between PM and nuclei, while KChIP2c has a more static distribution. Chronic hypertension/hypertrophy differentially impacts the expression of KChIP2a and 2c.