Pecerulean c1

T.C. Holmes (University of California, Irvine, CA) provided the rat Kv1.3 in a pRcCMV construct. The channel was subcloned into pEYFP-C1 and pECerulean-C1 (Clontech). All Kv1.3 mutants were generated in the pEYFP-Kv1.3 channel using a QuikChange site-directed mutagenesis kit (Agilent Technologies). pEYFP-Kv1.3CBD_less was subcloned into pECerulean-C1. For oocyte injection, Kv1.3 and Kv1.3CBD_less were subcloned into pcDNA3 and were placed under the control of a T7 promoter and then the cRNA was synthetized. J.R. Martens (University of Florida Medical School) provided the rat Caveolin 1 (Cav1) in pECerulean-C1. Cav1 was cloned into pcDNA3. The plasma membrane marker Akt-PH-pDsRed (pDsRed-tagged pleckstrin homology (PH) domain of Akt) was a kind gift of F. Viana (Universidad Miguel Hernández, Spain). The ER marker (pDsRed-ER) was obtained from Clontech. The mitochondrial marker (pmitoRFP) was constructed by fusing the mitochondrial transit sequence of the human isovaleryl coenzyme A dehydrogenase to the N-terminus of RFP (pDsRed1-N1, Clontech). Constructs were verified by sequencing.

Rat Kv1.3 in pRcCMV was provided by T.C. Holmes (University of California, Irvine, CA). Rat Kv1.1 and Kv1.4 in pGEM7 and human Kv1.5 in pBK constructs were subcloned into pEYFP-C1 and pECerulean-C1 (Clontech). Kv1.3/Kv1.5 chimeras were generated in the pEYFP-rKv1.3 and pEYFP-hKv1.5 channels by inserting BglII and EcoRI sites in the N- and C-terminal domains of the channels. Mutants were generated using the QuikChange site-directed mutagenesis kits (Stratagene). LoopBAD (BAD, Biotin acceptor domain) sequence was inserted in the first extracellular loop of pEYFP-Kv1.3 within a preexisting NruI site for rKv1.3LoopBAD construct. E. coli Biotin ligase containing construct pBtac_BirA was used as previously described54 (link). Rat Cav 1 into pECerulean-C1 was provided from J.R. Martens (University of Florida Medical School). Cav1 was inserted into pcDNA3 by digestion of Cav-pECerulean (HindIII-BamHI). Constructs were verified by sequencing.