LVA and HVA calcium channel modulation by kelch-like 1 protein (KLHL1) in cultured hippocampal neurons

PERISSINOTTI PP; KALIL J; MARTIN J; PIEDRAS-RENTERÍA ES

Washington, DC

Congreso; Society for Neuroscience; 2011

KLHL1 is an actin-binding protein constitutively expressed in the brain that regulates neuronal structure and function. In HEK-293 cells, KLHL1 modulates alpha 1H but not a1G channel function by increasing current density and the time constant of deactivation. We have shown that the effect of KLHL1 on alpha 1H is due to enhanced alpha 1H recycling to the plasma membrane, and requires the presence of polymerized actin. Here we studied the distribution of KLHL1 and a1H in cultured rat hippocampal neurons (which express all three CaV3 LVA proteins in the somata) using inmunocytochemistry. alpha 1H channel localization occurs mainly at the plasma membrane, and in the presence KLHL1, HEK-293 cells showed increased alpha 1H channel signal localization in the recycling endosome. Co-localization of alpha 1H and KLHL1 and of alpha 1H with the recycling endosome maker Rab11 can similarly be detected in hippocampal neurons, supporting our observations from the in vitro system. We also analyzed the effect of KLHL1 on voltage-gated calcium currents using patch clamp recording. Low- and high-voltage calcium currents (LHA, HVA) were characterized after 3, 4, 7, 8, 9 and 10 days in vitro (DIV). LVA current density increased with time of culture, and was significantly increased by 35% from 8-10 DIV compared to 3-7 DIV (-30 mV, ANOVA, p < 0.05; Duncan?s Method, p < 0.05); whereas no differences in HVA current density were observed (+ 10 mV, ANOVA, p > 0.0.5). We used a knockdown approach to determine if endogenous KLHL1 modulates calcium channel function. KLHL1shRNA/GFP adenovirus or GFP adenovirus (control) were transduced into hippocampal neurons at 4-6 DIV and calcium currents were recorded at 8-10 DIV. LVA current density was significantly lower in neurons infected with shRNAKLHL1/GFP (1.5 ± 0.1 pA/pF, n=5) compared to control (2.8 ± 0.3 pA/pF, n=5, t-test, p < 0.05). In addition, neurons infected with shRNAKLHL1-GFP displayed a faster deactivation time constant (2.8 ± 0.2 ms, n = 5) compared to control neurons (3.5 ± 0.2 ms, n = 5) (t-test, p < 0.05), consistent with the observed effects of KLHL1 on alpha1H in vitro. These results corroborate that KLHL1 is indeed a modulator of native LVA calcium channels in hippocampal neurons, most likely alpha1H (although the effect of KLHL1 on a1I channels remains to be elucidated). Supported by NSF 1022075.