Basic NeuroscienceQuantitative single-cell ion-channel gene expression profiling through an improved qRT-PCR technique combined with whole cell patch clamp
Introduction
Ion channels create gated transmembrane pores with unique gating kinetics that underlie cellular excitability (Hille, 2001). Each channel is composed of pore forming α-subunit(s) that can associate with other auxiliary subunits, which can modulate channel function or anchor the protein to cytoskeletal elements creating multi-subunit channel complexes. More than 100 different genes of α-subunits have been characterized (Catterall et al., 2005, Clapham et al., 2005, Gutman et al., 2005, Hofmann et al., 2005, Wei et al., 2005), which are further diversified by alternative splicing and post-translational modifications. This results in a wide variety of in vivo currents, presumably subserving the specific need of the various excitable tissues.
Obviously, this diversity complicates the characterization of the underlying molecular architecture of the channel complex (Fig. 1A). Even among neurons of a same type, large differences in the level of channel expression have been observed (Achard and De Schutter, 2006, Aptowicz et al., 2004, Mee et al., 2004, Schulz et al., 2006, Swensen and Bean, 2005). Furthermore, during development the expression profile of channel subunits may change substantially (Butler et al., 1998, Fitzakerley et al., 2000, Franco et al., 2001). The patch-clamp technique, a technique used to study the electrophysiology of excitable cells (Hamill et al., 1981), operates at the single cell level and allows exploration of cell-to-cell phenotypic variability. This variability can be the result of different mRNA transcript levels or originate from differences in post-translational modification such as e.g. phosphorylation. Therefore, analyzing both the transcriptome and the electrophysiological profile of the same cell could prove whether the observed variability finds its origin in the mRNA pool or not.
Most studies have combined patch-clamp experiments with end-point PCR and simplified variability to the presence or absence of expression (Audinat et al., 1996, Bochet et al., 1994, Chiang, 1998, Koizumi et al., 2004, Lambolez et al., 1992, Nissant et al., 2004, Toledo-Rodriguez et al., 2004). A few attempts have been made to combine single cell patch clamping with quantitative techniques measuring mRNA of the same cell, but they suffered from experimental noise making correlations between the electrophysiological data and mRNA transcript levels very difficult (Liss et al., 2001). This noise has multiple possible sources: variable extraction efficiency, differences in reverse transcription efficiency and the quantification by real-time PCR itself. Here we describe a refinement and optimization of the technique that combines whole cell path clamp analysis with quantitative real-time (qRT) PCR to determine the amount of a specific transcript at the single cell level. We applied it to the expression of Kv2.1 channels, a type of delayed rectifier potassium channel that regulates action potential duration and neuronal excitability (Blaine and Ribera, 1998, Misonou et al., 2005). In both stably transfected mouse Ltk− cells expressing Kv2.1 and mouse embryonic dorsal root ganglion neurons a good correlation between delayed rectifier current density and Kv2.1 transcript level was obtained, after proper normalization.
Section snippets
Electrophysiology
RNAse free patch pipettes were made from borosilicate glass capillaries with an outer diameter of 1.5 mm (Hilgenberg, Malsfeld, Germany) by baking the glass capillaries for at least 2 h at 200 °C after sonication in absolute alcohol. Pipettes with a resistance ranging from 2 to 4 MΩ were pulled on a horizontal P-87 puller (Sutter Instrument company, Novato, USA). Electrophysiological recordings were done at room temperature (21–23 °C) with an Alembic VE-2 Voltage clamp amplifier (Alembic Instruments
Whole cell patch clamp in combination with qRT-PCR
The RNA content of a single neuron is extremely low. On average a vertebrate neuron contains 50 pg of total RNA, 2% of which is mRNA. For a specific ion channel amounts of 0.2–2 fg of mRNA have been estimated, corresponding to a few tens to a few hundreds of actual molecules (Sucher et al., 2000). Consequently the cDNA yield (after reverse transcription of the isolated mRNA, see Section 2.3.1) is not sufficient to adhere to the general guidelines for performing qRT-PCR (Derveaux et al., 2010),
Discussion and conclusion
Using an optimized technique that combines whole cell patch-clamp recording with qRT-PCR we obtained a significant correlation between the NRQ of Kv2.1 and its current density using the phenotypic noise of an inducible promoter in Ltk− cells. Furthermore, implementing the same approach on embryonic DRG neurons yielded a strong correlation between Kv2 (ScTx sensitive delayed rectifier) current density and Kv2.1 mRNA expression level for only a limited data set. However, these correlations were
Acknowledgments
This work was supported by the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ grants FWO-G.0450.03 and FWO-G.0449.11, and a concerted research project Grant BOF-GOA 2004 from the University of Antwerp.
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These authors contributed equally to this work.