Cardiac-enriched LIM domain protein fhl2 is required to generate I(Ks) in a heterologous system.
OBJECTIVE
Co-expression of the KvLQT1 and minK potassium channel subunits is required to recapitulate I(Ks), the slow component of the cardiac delayed rectifier current, and mutations in either gene cause the congenital Long QT syndrome. It is becoming increasingly well-recognized that multiprotein channel complexes containing proteins capable of modulating channel function assemble at the plasma membrane. Thus, the aim of our study was to identify proteins involved in I(Ks) modulation.


RESULTS
Using a yeast-two-hybrid screen with the intracytoplasmic C-terminus of minK as bait, we identified the cardiac-enriched four-and-a-half LIM domain-containing protein (fhl2) as a potential minK partner. We show interaction between the two proteins in GST pulldown assays and demonstrate overlapping subcellular localization using immunocytochemistry of transfected cells supporting a potential interaction. At the functional level, expression of KvLQT1and minK in HEK cells, which lack endogenous fhl2 protein, generated I(Ks) only when fhl2 was co-expressed. By contrast, in CHO-K1 cells, which express fhl2 endogenously, I(Ks) was suppressed by anti-fhl2 antisense which did not affect the currents generated by KvLQT1alone.


CONCLUSIONS
These data indicate that at least in heterologous cells, the generation of I(Ks) requires fhl2 as an additional protein component.
Kupershmidt
Sabina
S
Department of Pharmacology, 561A PRB, Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA. sabina.kupershmidt@vanderbilt.edu
Yang
Iris C-H
IC
Sutherland
Margaret
M
Wells
K Sam
KS
Yang
Tao
T
Yang
Ping
P
Balser
Jeffrey R
JR
Roden
Dan M
DM
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D005818:Genetic Engineering; D018398:Homeodomain Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007150:Immunohistochemistry; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D008133:Long QT Syndrome / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection; D020798:Two-Hybrid System Techniques
D018398:Homeodomain Proteins; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D016376:Oligonucleotides, Antisense; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D014157:Transcription Factors; C059983:potassium channel protein I(sk)