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FRET analysis of KLHL1 homo‐dimerization and interaction with actin: influence of Ca V 3.2 calcium channel interaction with the KLHL1 complex
Author(s) -
Martin Hannah RS,
Sivakumaran Vidhya,
Benzow Kellie A,
Koob Michael D,
Martin Jody L,
Robia Seth L,
PiedrasRenteria Erika L
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.600.7
Subject(s) - förster resonance energy transfer , chemistry , actin , protein subunit , biophysics , acceptor , dimer , protein–protein interaction , fluorescence , biochemistry , biology , gene , physics , organic chemistry , quantum mechanics , condensed matter physics
Kelch‐like 1 protein (KLHL1) binds polymerized actin and modulates voltage‐gated calcium channel activity. This brain specific protein contains an actin‐binding domain, and a BTB/POZ domain, which is involved in protein‐protein interactions in dimer and heterodimer formation. We hypothesize the KLHL1‐actin complex further interacts with Ca V 3.2 T‐type channel, resulting in channel stabilization at the membrane and up‐regulation of function. The objective of this study is to measure both the homo‐dimerization of KLHL1 and its interaction with actin in the presence and absence of the pore‐forming alpha subunit of the Ca V 3.2 calcium channel (α 1H ). Fluorescence resonance energy transfer, FRET, was measured using both acceptor photo‐bleaching and 3‐cube methods (utilizing the donor, acceptor, and FRET channels) in naïve and stably expressing α 1H HEK293 cells. CFP‐tagged KLHL1 at either the N or C termini was used to assess KLHL1 interaction with N‐terminal YFP‐tagged KLHL1 or N‐terminal YFP‐actin. Interaction between N‐terminally tagged actin‐KLHL1 (N‐N) was stronger than pairs tagged in opposite ends (N‐C), as assessed by their FRET efficiency (29.4% vs. 20.8 %). The presence of Ca V 3.2 altered these interactions, resulting in lower FRET efficiency in N‐N pairs (18.2%) but no change between N‐C actin‐KLHL1 pairs (24.4%). Similarly, KLHL1‐KLHL1 dimerization was stronger between N‐N pairs (19.2%) compared to N‐C pairs (10.4%) in naïve cells. This interaction was also altered by the presence of the calcium channel, but interestingly, in this case, N‐C KLHL1 pairs elicited higher FRET efficiency when the channel was present (10.4% vs. 16.5%), whereas it resulted in similar FRET values in the N‐N configuration (19.2% and 21.4%). Overall, these results suggest that the KLHL1 dimer‐actin complex interactions are significantly altered upon the complex‐induced stabilization of T‐type channels at the plasma membrane. Support or Funding Information This material is based upon work supported by the National Science Foundation grant # 1022075. H.R.S.M. was the recipient of a summer undergraduate research fellowship from the Cell and Molecular Physiology Department.