Alignment of herg potassium channel with known structures of potassium channel Name: Prachi Sharma Superviser: Prof. Gert Vriend introduction

4. DISCUSSION

Our main goal was to align sequences for each family together. We also wanted to align sequences from different families together. Sequences for same family were aligned using Walign option of WHAT IF program. We tried to find conserved residues. To find important residues for the ion conduction we performed two types of alignments. In first kind of alignment we tried to align all sequences from same family together we looked at the conservation of residues. It was found that residues in selectivity filter were highly conserved. We also found that residues in the transmembrane helices were also conserved (http://www.cmbi.ru.nl/~psharma/index.html). For selectivity filter we found a conserved motif (T/SxxTxGxG).

We obtained profile for each family from family specific alignments. In second type of alignment all the profiles from different families were aligned together.

Second type of alignment was done between profiles obtained from alignment of sequences from same family. These profiles were so dissimilar from each other that it was simply impossible to align these profiles together. So these alignments were aligned according the conserved motifs like transmembrane helices and selectivity filter. For example we aligned Helix5 (helices before selectivity filter) from different families together, then we aligned selectivity filter from different families together. We also aligned Helix6 (transmembrane helices after selectivity filter). We found that lengths of loop joining Helix5 to Selectivity filter and Selectivity Filter to Helix6 were different in different families (http://www.cmbi.ru.nl/~psharma/index.html).

From the sequence alignment of sequences of selectivity filter from different potassium channel we found that GYG is always conserved except the second selectivity filter of TM4 potassium channel which has GLG (Fig9).

Helix just before the selectivity filter (Helix5) from all the different families were also aligned together. During aligning these sequence Entropy/Variability value of each residues in the sequences were also taken into account. Thus fine tuning in alignments were done according to the Entropy/Variability plot (Fig11).

In the Fig11.A alignments of helices before Pore has been shown. These alignments have been changed according to the entropy variability (Fig11.B)values of Glycine 18 in the helix before first pore of TM4. According to entropy/variability values Glycine 20 was aligned at the position of Glycine 18.

When helix5 , SF and Helix6 of hERG aligned with Kcsa and TM6 profile we found that in Helix6 (helix which is just after selectivity filter) residues R or K and residue G (shown in red color in Fig 14) are always conserved . However from Helix5 (helix which is just before selectivity filter)alignment we could not find any conserved residue.

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4. CONCLUSION

To build a homology model for all of the families of potassium channel we need alignments of all sequences from each family. As sequences are not identical it is impossible to align sequences together. Because of this difficulty, in this project we tried to align conserved sequences from conserved structures found in the potassium channel like transmembrane helices and Selectively Filter. We tried to improve are alignment using entropy/variability values for each residue position. Our proposed alignment is a hypothesis and it is required to be validated and proved. To make a final alignment following three steps are followed untill a hypothesis is proved.

We did analysis and based on our analysis we have proposed a hypothesis. In this case our alignments are our hypothesis. Next step in this project is to prove the hypothesis experimentally. After proving the hypothesis next step would be building a homology model for each of the Potassium channel family based on these alignments.

From alignment of hERG ,Kcsa and TM6 profile wecan conclude that in Helix5 residues R/K and residue G (shown in red color in Fig 14) are always conserved and seems to have important functional relevance. However from Helix5 (helix which is just before selectivity filter)alignment we could not reach any conclusion.

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