T-type current modulation by the actin-binding protein Kelch-like 1.
We report a novel form of modulation of T-type calcium currents carried out by the neuronal actin-binding protein (ABP) Kelch-like 1 (KLHL1). KLHL1 is a constitutive neuronal ABP localized to the soma and dendritic arbors; its genetic elimination in Purkinje neurons leads to dendritic atrophy and motor insufficiency. KLHL1 participates in neurite outgrowth and upregulates voltage-gated P/Q-type calcium channel function; here we investigated KLHL1's role as a modulator of low-voltage-gated calcium channels and determined the molecular mechanism of this modulation with electrophysiology and biochemistry. Coexpression of KLHL1 with Ca(V)3.1 or Ca(V)3.2 (alpha(1G) or alpha(1H) subunits) caused increases in T-type current density (35%) and calcium influx (75-83%) when carried out by alpha(1H) but not by alpha(1G). The association between KLHL1 and alpha(1H) was determined by immunoprecipitation and immunolocalization in brain membrane fractions and in vitro in HEK-293 cells. Noise analysis showed that neither alpha(1H) single-channel conductance nor open probability was altered by KLHL1, yet a significant increase in channel number was detected and further corroborated by Western blot analysis. KLHL1 also induced an increase in alpha(1H) current deactivation time (tau(deactivation)). Interestingly, the majority of KLHL1's effects were eliminated when the actin-binding motif (kelch) was removed, with the exception of the calcium influx increase during action potentials, indicating that KLHL1 interacts with alpha(1H) and actin and selectively regulates alpha(1H) function by increasing the number of alpha(1H) channels. This constitutes a novel regulatory mechanism of T-type calcium currents and supports the role of KLHL1 in the modulation of cellular excitability.
Aromolaran
K A
KA
Loyola University Chicago, Maywood, IL, USA.
Benzow
K A
KA
Cribbs
L L
LL
Koob
M D
MD
Piedras-Rentería
E S
ES
D000199:Actins / Q000378:metabolism*; D000200:Action Potentials; D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D015640:Ion Channel Gating; D007700:Kinetics; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D000199:Actins; C504883:Cacna1h protein, mouse; D020747:Calcium Channels, T-Type; C412617:KLHL1 protein, human; C514889:KLHL1 protein, mouse; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D002118:Calcium