Modulation of alpha 1h calcium channels by kelch-like 1 actin-binding protein

KALIL J; PERISSINOTTI PP; KEELING G; BRISEÑO S; PIEDRAS-RENTERÍA ES

Colorado

Congreso; FASEB meeting: Ion channel regulation; 2011

Kelch-like 1 (KLHL1) is an actin-binding protein (ABP) involved in the regulation of neuronal structure and function, including voltage-gated calcium channel up-regulation. KLHL1 KO mice present a progressive loss of post-synaptic structures, motor coordination and gait abnormalities. We have shown that KLHL1 up-regulates alpha 1H T-type channel function by an increase in calcium current density that results from an increase in the number of active channels at the plasma membrane due to enhanced recycling endosome activity, in a process that involves stabilization of the actin cytoskeleton. Additionally, KLHL1 exerts a direct effect on the channel that results in a slower time constant of deactivation. The two effects of KLHL1 on alpha1H require the presence of polymerized actin and are mainly mediated by the C-terminus actin-binding kelch domain, although the amino terminal region of the protein is sufficient to enable the direct interaction between KLHL1 and alpha1H. Here we present data on our study of KLHL1 function, including the subcellular localization of alpha1H in the presence of KLHL1, and functional studies of KLHL1 in hippocampal neuron cultures. The subcellualr distribution of KLHL1 was assayed in the recycling endosome, early endosome, lysosome and late endosome of HEK-293 cells using immunocytochemistry. The greatest localization of KLHL1 was in Rab11 positive compartments (recycling endosome, 30% ± 0.11 positive correlation, n= 9), compared to the other compartments, with the least localization in TIP 47 positive areas (late endosome, 1.7% ± 0.05 PC, n= 7). Interestingly, although most alpha1H channel localization was found at the plasma membrane, when the intracellular localization of alpha1H channel was probed in the presence and absence of KLHL1, cells transfected with KLHL1 showed increased T-type channel signal in the recycling endosome (4.8% ± 0.08 PC, n= 9) compared to controls (0.33% ± 0.03 PC, n= 7); TIP-47 was used as a negative control and did not change in the presence of KLHL1. These data confirm KLHL-1´s residence within sub-cellular compartments occurs preferentially in the recycling endosome, with decreasing localization at the early endosome, lysosome and late endosome. Alpha1H subunit localization increased by an order of magnitude at the recycling endosome in the presence of KLHL1; further supporting the hypothesis that KLHL1 induces increased alpha1H function due to increased recycling to the plasma membrane. Supported by NSF1022075.