Adriana G. Prat

Regulation of Epithelial Sodium Channels by Short Actin Filaments

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 α, β, γ-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 (Po) of α,β,γ-rENaC. 2) α,β,γ-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 α,β,γ-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of α,β,γ-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of α,β,γ-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of α,β,γ-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of α,β,γ-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 α,β,γ-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 α,β,γ-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.

Actin filament organization is required for proper cAMP-dependent activation of CFTR

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl- currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl- conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl- currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl-channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl- channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

Renal Epithelial Protein (Apx) Is an Actin Cytoskeleton-regulated Na+ Channel

Apx, the amphibian protein associated with renal amiloride-sensitive Na+ channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na+ channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban-Sohraby, S. (1987) J. Biol. Chem. 262, 10613-10618), has previously failed to generate amiloride-sensitive Na+ currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P. (1992) J. Cell Biol. 119, 1497-1506). Renal epithelial Na+ channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A. (1991) Am. J. Physiol. 261, C882-C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60-900% increase in amiloride-sensitive (Ki = 3 μM) Na+ currents. Single channel Na+ currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na+ channel of A6 cells, including a 6-7 pS single channel conductance and a perm-selectivity of Na+:K+ of 4:1. Na+ channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na+ reabsorption in the amphibian kidney.

Chapter 17 Role of Actin Filament Organization in Ion Channel Activity and Cell Volume Regulation

Publisher Summary This chapter describes the role of actin-filament organization on ion-channel activity and cell-volume regulation. Actin can be found in several different states within the intracellular compartment. Dynamic transitions entailing changes from one conformation to another may be central to such mechanically related events as cell locomotion and rounding, cytokinesis and spreading, and intracellular transport to the cell surface. Actin filaments may be also linked to osmotically induced phase transitions, which in turn may represent novel electroosmotic signaling events. These proteins therefore help maintain a balance between G- and F-actin concentrations. Another important family of ABPs, however, actually binds already formed actin filaments (F-actin). Most F-actin-binding proteins are known to have at least two actin-binding domains, thus allowing coupling to more than one actin filament. Depending on the distance between the actin-binding domains, these proteins may enable either the bundling of actin (tightly packed arrays of filaments), as observed in the complexes of F-actin found in microvilli, or the crosslinking of actin into 3-dimensional actin filament networks with gel behavior.