Membrane microdomain determines the specificity of receptor-mediated modulation of Kv7/M potassium currents

Highlights

• Disruption of lipid rafts reduces B₂ receptor-mediated Kv7/M current inhibition.

• B₂R and related signaling molecules, but not M₁R were found in the raft fractions in rat superior cervical ganglion.

• Disruption of the lipid rafts diminishes BK-induced intracellular Ca²⁺ rise.

• The lipid raft is essential for the coupling of IP₃R and B₂R.

Abstract

The Kv7/M current is one of the major mechanisms controlling neuronal excitability, which can be modulated by activation of the G protein-coupled receptor (GPCR) via distinct signaling pathways. Membrane microdomains known as lipid rafts have been implicated in the specificity of various cell signaling pathways. The aim of this study was to understand the role of lipid rafts in the specificity of Kv7/M current modulation by activation of GPCR. Methyl-β-cyclodextrin (MβCD), often used to disrupt the integrity of lipid rafts, significantly reduced the bradykinin receptor (B₂R)-induced but not muscarinic receptor (M₁R)-induced inhibition of the Kv7/M current. B₂R and related signaling molecules but not M₁R were found in caveolin-containing raft fractions of the rat superior cervical ganglia. Furthermore, activation of B₂R resulted in translocation of additional B₂R into the lipid rafts, which was not observed for the activation of M₁R. The increase of B₂R-induced intracellular Ca²⁺ was also greatly reduced after MβCD treatment. Finally, B₂R but not M₁R was found to interact with the IP₃ receptor. In conclusion, the present study implicates an important role for lipid rafts in mediating specificity for GPCR-mediated inhibition of the Kv7/M current.

Introduction

The M-type potassium channel plays a crucial role in controlling neuronal excitability, and its malfunction leads to diseases such as epilepsy (Marrion, 1997, Robbins, 2001, Wickenden et al., 2004, Surti and Jan, 2005). The channel is encoded by the Kv7/KCNQ potassium family, with heteromers composed most often of the Kv7.2 and Kv7.3 subunits, although other Kv7 members may also contribute (Wang et al., 1998, Passmore et al., 2003, Miceli et al., 2008).

The Kv7/M current can be modulated by many neuronal transmitters and peptides, and great achievements have been made toward understanding the underlying molecular mechanism(s) (Shapiro et al., 1994, Cruzblanca et al., 1998, Wallace et al., 2002, Gamper and Shapiro, 2003, Zhang et al., 2011). The most well-studied regulation of Kv7/M is mediated through G_q protein-coupled membrane receptors (G_qPCRs) involving two key signaling molecules, phosphatidylinositol 4,5-bisphosphates (PIP2) and intracellular Ca²⁺ (Yu et al., 1994, Brown et al., 2007, Hughes et al., 2007). While the activation of some G_qPCRs hydrolyze and reduce membrane PIP2 (Shapiro et al., 1994, Marrion, 1997, Bofill-Cardona et al., 2000, Winks et al., 2005, Brown et al., 2007, Zhang et al., 2011), activation of other G_qPCRs increase intracellular Ca²⁺ levels (Cruzblanca et al., 1998, Bofill-Cardona et al., 2000, Brown et al., 2007, Zaika et al., 2007). Thus, different G_qPCRs can confer specificity/selectivity for each pathway, but both leading to the inhibition of Kv7/M channel function. A receptor-dependent microdomain coupling between the receptor and the IP₃ receptor (Delmas et al., 2002), and the intracellular Ca²⁺-dependent feed-back re-synthesis of membrane PIP2 (Gamper et al., 2004, Winks et al., 2005, Loew, 2007) have been suggested as specific mechanisms of G_qPCR modulation of the Kv7/M current.

However, a general principle for the specificity of receptor-mediated modulation of Kv7/M function is still lacking. It is clear that while the specificity exists with native receptors in neuronal cells, it disappears when the receptors are overexpressed. For example, activation of the bradykinin receptor (B₂R) in superior cervical ganglion (SCG) neurons and dorsal root ganglion (DRG) neurons increases intracellular Ca²⁺ but does not reduce the membrane PIP2 level (Hughes et al., 2007); meanwhile, activation of the B₂ receptor overexpressed in CHO cells reduces the membrane PIP2 level (Liu et al., 2010). Similarly, activation of the muscarinic receptor (MR) does not increase intracellular Ca²⁺ in SCG neurons (Delmas et al., 2002), but increases intracellular Ca²⁺ significantly in CHO cells where MR receptors are overexpressed (Zhang et al., 2003). These observations suggest that a delicate and complex system exists to ensure the specificity. One possible explanation is that specific spatial organization and dynamics in the receptor-mediated systems may coordinate the specificity of these signaling events. In this regard, the lipid raft may serve as a platform for these complex and specific signaling pathways. The lipid raft is a microdomain structure on the cell membrane, ranging in size from 55 to 300 nm, and is enriched with cholesterol and sphingomyelin (Fantini et al., 2002, Radeva and Sharom, 2004, Shimada et al., 2005). As lipid rafts are characterized by a low-buoyant density (Persaud-Sawin et al., 2009) and detergent insolubility (Weerth et al., 2007), they are also known as detergent-resistant membranes (DRM). Signaling molecules concentrated in these rafts have been shown to determine the specificity of various cell signaling pathways (Tsui-Pierchala et al., 2002, Calaghan et al., 2008, Patel et al., 2008, Morenilla-Palao et al., 2009, Pani and Singh, 2009).

In this study, we hypothesized that lipid rafts may play a crucial role in the specificity of receptor-mediated modulation of the Kv7/M current. To test this notion, we prepared the microdomain membranes with characteristics of lipid rafts, as indicated by the presence of caveolin from SCGs using sucrose density gradient separation. The fractions were analyzed for proteins involved in Kv7/M channel modulation as well as caveolin-1, a marker for lipid rafts (Hill et al., 2008, Liu and Pilch, 2008). We found that a number of signaling proteins associated with Kv7/M channel modulation were present in the caveolin-1 rich fractions. Consistent with our hypothesis, the results suggested that the specificity of Kv7/M channel modulation was sensitive to the spatial integrity of lipid rafts.