Regulation of Epithelial Sodium Channels by Short Actin Filaments

Abstract

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the ␣, ␤, ␥-rENaC (rat epithelial Na ؉ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (P o ) of ␣,␤,␥-rENaC. 2) ␣,␤,␥-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of ␣,␤,␥-rENaC, although with a shortened time course and with a lower magnitude of change in P o . 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of ␣,␤,␥-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of ␣,␤,␥-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of ␣,␤,␥-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of ␣,␤,␥-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate ␣,␤,␥-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned ␣,␤,␥-rENaCs form a core conduction unit of epithelial Na ؉ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.Membrane transport protein-cytoskeleton interactions are thought to be important not only for restricting various transporters to specific membrane domains, especially in epithelia, but also for regulating transport activity (1, 2). Actin networks interact with the plasma membrane either by direct binding (3, 4) or through anchoring proteins, including actin-binding protein (filamin), spectrin (fodrin, the non-erythroid form of spectrin), and ankyrin (5-8). The actin cytoskeleton has been shown to interact with a variety of transmembrane proteins, including ion transport molecules such as the band 3 anion exchanger (9), the epithelial Na ϩ /K ϩ -ATPase (6, 10), rat brain voltage-sensitive Na ϩ channels (11, 12), the Na ϩ -K ϩ -Cl Ϫ cotransporter (13), and the Na ϩ -H ϩ exchanger (14). Recently, functional interactions between the actin cytoskeleton and ion channels have been demonstrated for Na ϩ channel activity in epithelial cells (15), N-methyl-D-aspartic acid-activated channels in neurons (16), the Na ϩ /K ϩ -ATPase (17), a renal K ϩ channel (18), and two epithelial anion channels (namely, a renal Cl Ϫ channel (19) and the cystic fibrosis transmembrane conductance regulator (20, 21)). Apically located, renal amiloride-sensitive Na ϩ channels have also been shown to be associated directly with the actin-based membrane cytoskeleton (15, 22). Moreov...

Distinct proteomic profiles of amphetamine self-administration transitional states

“…ARP3 and Coronin 1A, both of which increase actin filament assembly, 30,31 are decreased specifically during abstinence. Taken together, this would suggest a decrease in actin polymerization during abstinence which could alter potassium, 32 sodium, 33 calcium, AMPA, 34 and NMDA receptor channel function. 35 Owing to its regulation of receptor functionality, actin polymerization regulation can affect the potentiation or depression of a neuron.…”

The Actin-Severing Protein Gelsolin Modulates Calcium Channel and NMDA Receptor Activities and Vulnerability to Excitotoxicity in Hippocampal Neurons

“…Some data suggest that actin may interact directly with ion channels. For example, short actin filaments enhanced sodium channel activity in planar lipid bilayers, and cytochalasin D blocked the effect of the actin filaments (Berdiev et al, 1996). On the other hand, there are a number of actin-binding proteins that link actin with membranes, and some such proteins (e.g., dystrophin) have been implicated in modulating calcium influx (for review, see Hartwig, 1994).…”

“…Recent findings suggest that, in addition to their roles in regulating cell structure and motility, actin filaments may f unction in the modulation of ion channel f unction. The evidence is based on pharmacological data showing that the actin filament-disrupting agent cytochalasin D can alter ion flux through Na ϩ channels (C antiello et al, 1991;Undrovinas et al, 1995;Berdiev et al, 1996), voltage-dependent calcium channels (VDCC) (Johnson and Byerly, 1993), and NMDA receptor channels (Rosenmund and Westbrook, 1993). Whether actin normally plays roles in regulating Ca 2ϩ influx in physiological and pathophysiological settings is unknown.…”

ATP masks stretch activation of epithelial sodium channels in A6 distal nephron cells

“…Nonetheless, the mechanosensitivity of ENaC remains a matter of controversy despite a variety of studies attempting to address this issue. Examples of studies in which ENaC appears to be stretch sensitive include the observations that the purified renal ENaC in planar lipid bilayer membranes can be activated by hydrostatic pressure (1,4,8) and that expression of ␣-ENaC in mammalian cells results in stretch-activated channel activity (10). Recent studies have shown that osmotic stretch regulates rat ␣-, ␤-, and ␥-ENaC expressed in Xenopus laevis oocytes (9).…”