Aplicación del Pushover a marcos de concreto reforzado mediante el programa del Sap

KCNE2 contains one extracellular N-terminal that includes two predicted N-glycosylation sites (N6 and N29) (Figure 1.10 and Figure 1.12) (Zhang et al. 2012), one transmembrane domain and one intracellular C-terminal domain that includes protein kinase C-mediated (PKC) consensus phosphorylation sites (T71 and S74; Figure 1.10) (Abbott 1999; McCrossan and Abbott 2004). This small integral membrane subunit has been shown to co-assemble with HERG - the KCNH2 gene product - in order to form a functional unit of the IKr channel (Abbott 1999). However, in a

subsequent study by Jiang and colleagues it was found that a very small amount of KCNE2 showed association with HERG in co-immunoprecipitation analysis (Jiang et al. 2009).

This gave rise to the suggestion that this specific protein has other possible interacting partners (Jiang et al. 2004). Other investigators suggested that KCNE2 might interact/associate with KCNQ1 and KCNQ3 and draw out a voltage-dependent current. Or it may also associate with Hyperpolarization Activated Cyclic Nucleotide-Gated Potassium Channel 1 and 2 (HCN1and HCN2) to increase the potassium current (Yu et al. 2001).

The association of KCNE proteins with the appropriate α-subunits have been found to take place in the endoplasmic reticulum (ER) (Chandrasekhar et al. 2006) as well as at the plasma membrane (Jiang et al. 2009) with more than one protein co-assembling with the channel

31 complex. Evidence suggests that one of the KCNE proteins could take the place of another family member to influence the current properties (Eldstrom and Fedida 2011).

In a 2008 study, Abbott and colleagues determined the secondary structure of KCNE2 as largely α-helical and concluded that the predicted transmembrane and intracellular domains require an extensive hydrophobic interaction in order for the molecule to take on an ordered, non- aggregated structure (Abbott et al. 2008).

The N-terminal domain was found to be the only water-soluble domain in this study which indicated the consistency with the predicted extracellular location of this section. Abbott and co- workers mention that the function of the N-terminal of KCNE2 has not yet been elucidated but the removal of residues four-nine from the N-terminal of KCNE1 results in the loss of channel function. This might be extrapolated to KCNE2 due to the homology shared between these two subunits (Abbott et al. 2008).

Additionally two inherited mutations (Q9E and T8A) located in the N-terminal of KCNE2 (Figure 1.12) have been associated with acquired (drug-induced) forms of arrhythmias (Abbott 1999). One of these mutations - Q9E - was found to increase the sensitivity of IKr channel

blockage for drugs such as clarithromycin. This complicates the repolarization action of mutant IKr complexes and is believed to contribute to the prolongation of the QTc interval of the cardiac

action potential; regardless of the presence of drugs (Abbott 1999).

The transmembrane domain structure of KCNE1 has proven to be more difficult to determine than anticipated; with several studies suggesting controversial conformations for this domain. Some investigators proposed a β-conformation, while others concluded an α-helical structure for this section of the protein (Abbott et al. 2008).

Similarly, the transmembrane domain of KCNE2 presented with some difficulty but was ultimately identified through infrared spectroscopy as predominantly α-helical with a minor intra-molecular β-strand conformation (Abbott et al. 2008).

The intracellular C-terminal domain of KCNE2 was shown to be insoluble in water, indicating that a hydrophobic interaction is essential for solubility as well as an ordered secondary structure of this domain (Abbott et al. 2008).

32 Exon 1 1 GTAAGGTGAAGGTGCCCAGCAGGCTGAGGCTTGTGTGCAACCCAGAAGAGAGCTCGCTAA ... 61 CGCCAGCAAGAAGGTTCAGAACAGCCTGGCTTTGGAAAGGAATTTCATCCTGCCCACACA ... Exon 2 N-Terminal

121 CTGCATAGCAGGAGGGAAGCATGTCTACTTTATCCAATTTCACACAGACGCTGGAAGACG ...-M--S--T--L--S--N--F--T--Q--T--L--E--D-- 181 TCTTCCGAAGGATTTTTATTACTTATATGGACAATTGGCGCCAGAACACAACAGCTGAGC 14 V--F--R--R--I--F--I--T--Y--M--D--N--W--R--Q--N--T--T--A--E— Transmembrane domain 241 AAGAGGCCCTCCAAGCCAAAGTTGATGCTGAGAACTTCTACTATGTCATCCTGTACCTCA 34 Q--E--A--L--Q--A--K--V--D--A--E--N--F--Y--Y--V--I--L--Y--L-- C-Terminal 301 TGGTGATGATTGGAATGTTCTCTTTCATCATCGTGGCCATCCTGGTGAGCACTGTGAAAT 54 M--V--M--I--G--M--F--S--F--I--I--V--A--I--L--V--S--T--V--K-- 361 CCAAGAGACGGGAACACTCCAATGACCCCTACCACCAGTACATTGTAGAGGACTGGCAGG 74 S--K--R--R--E--H--S--N--D--P--Y--H--Q--Y--I--V--E--D--W--Q-- 421 AAAAGTACAAGAGCCAAATCTTGAATCTAGAAGAATCGAAGGCCACCATCCATGAGAACA 94 E--K--Y--K--S--Q--I--L--N--L--E--E--S--K--A--T--I--H--E--N--

481 TTGGTGCGGCTGGGTTCAAAATGTCCCCCTGATAAGGGAGAAAGGCACCAAGCTAACATC 114 I--G--A--A--G--F--K--M--S--P--*-... 541 TGACGTCCAGACATGAAGAGATGCCAGTGCCACGAGGCAAATCCAAATTGTCTTTGCTTA ... 601 GAAGAAAGTGAGTTCCTTGCTCTCTGTTGAGAATTTTCATGGAGATTATGTGGTTGGCCA ... 661 ATAAAGATAGATGACATTTCAATCTCAGTGATTTATGCTTGCTTGTTGGAGCAATATTTT ... 721 GTGCTGAAGACCTCTTTTACTTTCCGGGCAAGTGAATGTCATTTTAATCAATATCAATGA ... 781 TGAAAATAAAGCCAAATTTGAAG ...

Figure 1.10: KCNE2 genomic and protein sequence. The sequence in orange marks the beginning of the second exon. The red codons, ATG and TGA, resembles the start and stop codons respectively. The sequences in blue,

purple and green indicate the N-terminus, the transmembrane domain and the C-terminus respectively. The section highlighted in light-blue in the protein sequence indicates the two glycosylation sites whereas the grey shaded area resembles the two PKC sites. Adapted from Eldtorm 2011and www.ensembl.org.

33 Finally Abbott and co-workers concluded that, although their findings were consistent with previous studies, it should be noted that KCNE2 is glycosylated at two sites in the N-terminal domain (Figure 1.10 and Figure 1.12) as well at the PKC sites in the C-terminal domain (Figure 1.10) and that this, in conjunction with KCNE2 interaction with α-subunits, might alter the structure of the KCNE2 protein (Abbott et al. 2008).