The secret life of CFTR as a calcium-activated chloride channel

Abstract

cAMP-stimulated anion conductance is defective in cystic fibrosis (CF). The regulatory domain of CFTR, the anion channel protein encoded by the CF gene, possesses an unusually high density of consensus sequences for phosphorylation by protein kinase A (14 in a stretch of <200 amino acids). Thus it is not surprising that CFTR is viewed primarily as a cAMP-stimulated anion channel, and most studies have focused on this mode of activation. However, there is growing evidence that CFTR also responds to Ca(2+)-mobilizing secretagogues and contributes substantially to cholinergic and purinergic responses in native tissues. G protein-coupled receptors that signal through Gαq can stimulate CFTR channels by activating Ca(2+)-dependent adenylyl cyclase and tyrosine kinases, and also by inhibiting protein phosphatase type 2A. Here we review evidence for these novel mechanisms of CFTR activation and discuss how they may help explain previous observations.

Figure 1. Time course and current–voltage relations of CFTR activation by carbachol and their dependence on intracellular Ca²⁺

A, representative whole cell currents measured in BHK cells made to co-express the muscarinic type 3 receptor and CFTR. Cells were exposed sequentially to carbachol (Cch), forskolin (Fsk) and CFTR_inh172, each at 10 μmol l⁻¹. Experiments were performed with or without 60 min preincubation with the membrane-permeant Ca²⁺ buffer BAPTA-AM (500 μmol l⁻¹) in the bath and pipette solutions. Note that stimulation was more robust with carbachol than with forskolin, that BAPTA-AM inhibited the Cch response >60%, and the CFTR inhibitor CFTRinh172 virtually abolished the response to Cch + Fsk. B, current–voltage relationship for currents measured in A during the plateau phase of stimulation.

Figure 2. Simplified scheme for Ca²⁺ activation of CFTR via PKA and tyrosine kinases

VIP receptors couple strongly to G_αs and weakly to G_αq, which stimulate adenylyl cyclase (AC) and phospholipase C (PLC), respectively. The muscarinic type 3 receptor (M3R) couples strongly to G_αq. cAMP activates CFTR channels by means of the canonical PKA pathway. Diacylglycerol and inositol trisphosphate (IP₃) activate protein kinase C (PKC) and mobilize Ca²⁺, which in turn activate the Pyk2/Src complex. Src stimulates CFTR activity by phosphorylating it directly and also by inhibiting its dephosphorylation through inactivation of protein phosphatase type 2A (PP2A). Under basal conditions, constitutive Ca²⁺ entry through store-operated Ca²⁺ channels partially activates adenylyl cyclase and induces tonic CFTR activity.