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cAMP-independent regulation of CFTR by the actin cytoskeleton
A G Prat1, Y F Xiao, D A Ausiello
1Renal Unit, Massachusetts General Hospital East, Charlestown 02129, USA.
Abstract:
Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.
Insights
Actin filaments regulate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Disruption of actin cytoskeleton activates CFTR, while actin addition restores PKA-dependent activation, indicating a crucial role for actin in CFTR function.
Area of Science:
- Cell Biology
- Ion Channel Physiology
- Molecular Medicine
Background:
- Protein kinase A (PKA) activation of epithelial sodium channels (ENaC) is known to require actin filaments.
- The role of the actin cytoskeleton in regulating other ion channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), remains less understood.
Purpose of the Study:
- To investigate whether the actin cytoskeleton modulates the activity of the human cystic fibrosis transmembrane conductance regulator (CFTR).
- To determine the mechanism by which actin filaments influence CFTR channel function and its regulation by PKA.
Main Methods:
- Utilized mouse mammary adenocarcinoma cells expressing either human CFTR or mock transfectants.
- Employed cytochalasin D (CD) to disrupt actin filaments and assessed whole-cell currents.
- Investigated the effect of adding purified actin to the cytosolic side of excised inside-out patches.
- Characterized the biophysical properties of actin-activated CFTR channels and tested inhibitors.
Main Results:
- Cytochalasin D (CD) treatment readily activated whole-cell currents in CFTR-expressing cells, but not in mock-transfected cells.
- Addition of actin to the cytosolic side of excised patches activated CFTR channels, indicating a direct regulatory role.
- Actin-activated CFTR channels exhibited specific conductance and were inhibited by known CFTR inhibitors and actin-binding proteins.
- Prolonged CD treatment inhibited CFTR activation by PKA agonists, but this inhibition was reversible upon subsequent actin addition.
Conclusions:
- CFTR channel activity is directly regulated by the actin cytoskeleton.
- Actin filaments play a critical role in both the basal and PKA-dependent activation of CFTR.
- These findings reveal a novel mechanism of CFTR regulation involving the actin cytoskeleton, potentially linking it to cellular mechanical properties and PKA signaling pathways.