Endogenous potassium channel ancillary subunits in heterologous expression systems
One of the primary tools for studying ion channel function is heterologous expression. After identification and cloning of an ion channel gene, the complementary DNA or RNA encoding the ion channel protein can be introduced into a cell to facilitate functional studies using, for example, patch clamp or two-electrode voltage clamp electrophysiology. The preferred features of cells used for heterologous expression studies of ion channels vary depending upon the goal of the particular study. If a 'native' environment is desired, a cell type mimicking the cell type in which the channel is expressed in vivo might be utilized. For many studies, it is more desirable that the cell is relatively 'electrically silent' until introduction of the cloned ion channel, so that the majority of the current under study arises from the introduced channel.
We often utilize the highly popular Xenopus laevis expression system for heterologous expression of ion channels for functional analysis using two-electrode voltage clamp. The Xenopus system facilitates highly robust expression of cloned channels, and is particularly useful for studies involving long pulses at highly polarized voltages, or for multiple recordings from the same cell over long periods of time. After observing anomalous results when recording currents generated by low-level heterologous expression of potassium channels in Xenopus oocytes, we examined a possible role for endogenous Xenopus potassium channel regulatory subunits. We first cloned a family of Xenopus MiRPs - single transmembrane domain potassium channel ancillary subunits that form complexes with potassium channel alpha subunits to alter their function. Next, using RNA interference (RNAi) to suppress endogenous MiRP expression, we showed that endogenous MiRPs are at sufficiently high native expression levels to influence functional studies of mammalian potassium channels in Xenopus oocytes, particularly at low heterologous expression levels such as those that might be used for single-channel studies.
Going forward, we intend to exploit the endogenous Xenopus oocyte MiRPs as a screening tool to better understand native regulation of potassium channel function, and use knowledge of the effects of endogenous MiRPs to enhance future structure-function studies employing the Xenopus oocyte system.
Relevant articles
Gordon, E., Roepke, T.K., & Abbott G.W. Endogenous MinK-Related Peptides Govern the Activation Kinetics of Kv2.1 Potassium Channels in Xenopus oocyte studies. Biophysical Journal 90(4):1223-31, 2006
Gordon E & Abbott G.W. RNAi in Xenopus laevis oocytes in Gene Silencing by RNA Interference: Technology and Application CRC Press, FL, U.S.A., 2004
Anantharam A, Lewis A, Panaghie G, Gordon E, McCrossan ZA, Lerner DJ, Abbott G.W. RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K channel function in oocyte expression studies. J Biol Chem. 278(14):11739-45, 2003.
