Amiloride-sensitive sodium channel is linked to the cytoskeleton in renal epithelial cells.

Abstract

Amiloride-sensitive sodium channels are localized to the microvillar domain of apical membranes in sodium-transporting renal epithelial cells. To elucidate the elements that maintain sodium channel distribution at the apical membrane, we searched for specific proteins associating with the channel. Triton X-100 extraction of A6 epithelial cells reveals that sodium channels are associated with detergentinsoluble and assembled cytoskeleton. Indirect immunofluorescence and confocAl microscopy show that sodium channels are segregated to the apical microvillar membrane and colocalize with ankyrin, fodrin, and actin. We document by immunoblot analysis that ankyrin and fodrin remain associated with sodium channels after isolation and purification from bovine renal papillae. 1MI-abeled ankyrin can be precipitated by anti-sodium-channel antibodies only in the presence of purified bovine sodium-channel complex. Direct binding of '2I-labeled ankyrin shows ankyrin binds to the 150-kDa subunit of the channel. Fluorescence photobleach lateral-diffusion measurements indicate sodium channels are severely restricted in their lateral mobility. We conclude that ankyrin links the amioride-sensitive sodium channel to the underlying cytoskeleton and this association may sequester sodium channels at apical microvilli and maintain their polarized distribution in renal epithelial cells.protein spectrin (4, 7-9), thus forming an integral membrane protein-ankyrin-spectrin-actin complex. To date, ankyrin and spectrin have been shown to associate with the anion exchanger (band 3) of erythrocytes, voltage-dependent Na' channels of neurons, lymphocyte adhesion antigen Pgp-1, ankyrin-binding glycoprotein 205 from brain, Na+/K+-

Ankyrin–Tiam1 Interaction Promotes Rac1 Signaling and Metastatic Breast Tumor Cell Invasion and Migration

“…These molecules belong to a family of related genes that probably arose by duplication and divergence of a common ancestral gene. Ankyrin is known to bind to a number of plasma membrane–associated proteins including the following: band 3, two other members of the anion exchange gene family (Bennet, 1992), Na + /K + -ATPase (Nelson and Veshnock 1987; Zhang et al 1998), the amiloride-sensitive Na + channel (Smith et al 1991), the voltage-dependent Na + channel (Kordeli et al 1995), Ca 2+ channels (Bourguignon et al 1993b, Bourguignon et al 1995a; Bourguignon and Jin 1995) and the adhesion molecule CD44 (Bourguignon et al 1986, Bourguignon et al 1991, Bourguignon et al 1992, Bourguignon et al 1993a; Kalomiris and Bourguignon 1988, Kalomiris and Bourguignon 1989; Lokeshwar and Bourguignon 1991, Lokeshwar and Bourguignon 1992; Lokeshwar et al 1994, Lokeshwar et al 1996). It has been suggested that the binding of ankyrin to certain membrane-associated molecules is necessary for signal transduction, cell adhesion, membrane transport, cell growth, migration, and tumor metastasis (Bennet, 1992; Bourguignon et al 1995b, 1996, Bourguignon et al 1997, Bourguignon et al 1998a; De Matteis and Morrow 1998; Zhu and Bourguignon 1998, Zhu and Bourguignon 2000).…”

Epithelial Na+ Channels Are Activated by Laminar Shear Stress

“…Although our experiments suggest that ENaC is a mechanosensitive channel, mechanisms by which mechanical forces modulate ENaC gating have not been defined. Previous studies have suggested that ENaC is tethered to the cytoskeleton (30,31). Although evidence does not exist for interactions between ENaC extracellular domains and the extracellular matrix or other extracellular proteins, approximately two-thirds of the mass of each Na ϩ channel subunit is present within the extracellular space.…”

Cloning and Functional Expression of Human Short TRP7, a Candidate Protein for Store-operated Ca2+ Influx

“…It is possible that the presence of a Pro instead of a Leu in this position may affect the structure of the hTRP7 N-terminal region and influence both the interaction of the channel with the cytoskeleton and the function of the channel itself (53,55,(57)(58)(59)(60). Future studies using mutagenesis, functional analysis, and yeast two-hybrid screening will help to address the importance of this amino acid substitution.…”