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A functional Kv1.2-hERG chimaeric channel expressed in Pichia pastoris.

Mandeep S Dhillon, Christopher J Cockcroft, Tim Munsey, Kathrine J Smith, Andrew J Powell, Paul Carter, David C Wrighton, Hong-lin Rong, Shahnaz P Yusaf, Asipu Sivaprasadarao

Sci Rep, 2014 , 4, 4201

Members of the six-transmembrane segment family of ion channels share a common structural design. However, there are sequence differences between the members that confer distinct biophysical properties on individual channels. Currently, we do not have 3D structures for all members of the family to help explain the molecular basis for the differences in their biophysical properties and pharmacology. This is due to low-level expression of many members in native or heterologous systems. One exception is rat Kv1.2 which has been overexpressed in Pichia pastoris and crystallised. Here, we tested chimaeras of rat Kv1.2 with the hERG channel for function in Xenopus oocytes and for overexpression in Pichia. Chimaera containing the S1-S6 transmembrane region of HERG showed functional and pharmacological properties similar to hERG and could be overexpressed and purified from Pichia. Our results demonstrate that rat Kv1.2 could serve as a surrogate to express difficult-to-overexpress members of the six-transmembrane segment channel family.

http://www.ncbi.nlm.nih.gov/pubmed/24569544