Part:BBa_K2263000

Part K2263000 is a mammalian membrane protein gene, and is interesting for projects in the field of electrophysiology.

Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are a family of ion channels responsible for the generation of rhythmic activity in heart and nerve cells. Consequently, they are often nicknamed pacemaker channels. They show several unique characteristics: first of all, the channels are opened in response to hyperpolarisation instead of depolarisation. Furthermore, they are not completely selective for K+-ions, and as a result, there is a small Na+ inward current which causes a slow depolarisation of the membrane, the so-called funny current or pacemaker depolarisation. (1) These attributes cause the particular rhythmic behaviour of cells containing HCN channels.

In our project, we used mHCN2 to generate a stable electrophysiological oscillation in HEK cells. (Read more about that on our wiki: http://2017.igem.org/Team:KU_Leuven ) In this project, we used the mHCN2 gene encoded by the wildtype sequence, and not the biobrick-compatible version we present here. However, we have made a great effort to introduce the silent mutations in a smart and codon-optimising way, and we hope other teams will use and characterise this part in the future.

For teams that want to use this biobrick, we suggest to clone the part into a mammalian expression vector, followed by an internal ribosome entry site (IRES) and a fluorescent marker. In this case, the part and the marker are transcribed at the same time, but translated independently. In such a system, the marker indicates that the part is transcribed in the cell, but they are not physically linked. This is beneficial, as a linked marker protein may affect or hinder the protein function.

IRES sequences and fluorescent markers can be found throughout the registry. Furthermore, many labs using mammalian expression vectors will own empty vectors containing an IRES and a marker. You could clone this part directly into such a vector.

(1) Kaupp UB, Seifert R. Molecular diversity of pacemaker ion channels (2001) Annu Rev Physiol. 63:235-57.