Functional coupling of TRPV4 cationic channel and large conductance, calcium-dependent potassium channel in human bronchial epithelial cell lines.

Calcium-dependent potassium channels are implicated in electrolyte transport, cell volume regulation and mechanical responses in epithelia, although the pathways for calcium entry and their coupling to the activation of potassium channels are not fully understood. We now show molecular evidence for the presence of TRPV4, a calcium permeable channel sensitive to osmotic and mechanical stress, and its functional coupling to the large conductance calcium-dependent potassium channel (BK(Ca)) in a human bronchial epithelial cell line (HBE). Reverse transcriptase polymerase chain reaction, intracellular calcium imaging and whole-cell patch-clamp experiments using HBE cells demonstrated the presence of TRPV4 messenger and Ca(2+) entry, and outwardly rectifying cationic currents elicited by the TRPV4 specific activator 4alpha-phorbol 12,13-didecanoate (4alphaPDD). Cell-attached and whole-cell patch-clamp of HBE cells exposed to 4alphaPDD, and hypotonic and high-viscosity solutions (related to mechanical stress) revealed the activation of BK(Ca) channels subsequent to extracellular Ca(2+) influx via TRPV4, an effect lost upon antisense-mediated knock-down of TRPV4. Further analysis of BK(Ca) modulation after TRPV4 activation showed that the Ca(2+) signal can be generated away from the BK(Ca) location at the plasma membrane, and it is not mediated by intracellular Ca(2+) release via ryanodine receptors. Finally, we have shown that, unlike the reported disengagement of TRPV4 and BK(Ca) in response to hypotonic solutions, cystic fibrosis bronchial epithelial cells (CFBE) preserve the functional coupling of TRPV4 and BK(Ca) in response to high-viscous solutions.

Human TRPV4 channel splice variants revealed a key role of ankyrin domains in multimerization and trafficking.

The TRPV4 cation channel exhibits a topology consisting of six predicted transmembrane domains (TM) with a putative pore loop between TM5 and TM6 and intracellular N- and C-tails, the former containing at least three ankyrin domains. Functional transient receptor potential (TRP) channels are supposed to result following the assembly of four subunits. However, the rules governing subunit assembly and protein domains implied in this process are only starting to emerge. The ankyrin, TM, and the C-tail domains have been identified as important determinants of the oligomerization process. We now describe the maturation and oligomerization of five splice variants of the TRPV4 channel. The already known TRPV4-A and TRPV4-B (delta384-444) variants and the new TRPV4-C (delta237-284), TRPV4-D (delta27-61), and TRPV4-E (delta237-284 and delta384-444) variants. All alternative spliced variants involved deletions in the cytoplasmic N-terminal region, affecting (except for TRPV4-D) the ankyrin domains. Subcellular localization, fluorescence resonance energy transfer, co-immunoprecipitation, glycosylation profile, and functional analysis of these variants permitted us to group them into two classes: group I (TRPV4-A and TRPV4-D) and group II (TRPV4-B, TRPV4-C, and TRPV4-E). Group I, unlike group II variants, were correctly processed, homo- and heteromultimerized in the endoplasmic reticulum, and were targeted to the plasma membrane where they responded to typical TRPV4 stimuli. Our results suggest that: 1) TRPV4 biogenesis involves core glycosylation and oligomerization in the endoplasmic reticulum followed by transfer to the Golgi apparatus for subsequent maturation; 2) ankyrin domains are necessary for oligomerization of TRPV4; and 3) lack of TRPV4 oligomerization determines its accumulation in the endoplasmic reticulum.

TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity.

Autoregulation of the ciliary beat frequency (CBF) has been proposed as the mechanism used by epithelial ciliated cells to maintain the CBF and prevent the collapse of mucociliary transport under conditions of varying mucus viscosity. Despite the relevance of this regulatory response to the pathophysiology of airways and reproductive tract, the underlying cellular and molecular aspects remain unknown. Hamster oviductal ciliated cells express the transient receptor potential vanilloid 4 (TRPV4) channel, which is activated by increased viscous load involving a phospholipase A(2)-dependent pathway. TRPV4-transfected HeLa cells also increased their cationic currents in response to high viscous load. This mechanical activation is prevented in native ciliated cells loaded with a TRPV4 antibody. Application of the TRPV4 synthetic ligand 4alpha-phorbol 12,13-didecanoate increased cationic currents, intracellular Ca(2+), and the CBF in the absence of a viscous load. Therefore, TRPV4 emerges as a candidate to participate in the coupling of fluid viscosity changes to the generation of the Ca(2+) signal required for the autoregulation of CBF.

Swelling-activated Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia.

The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca(2+) signal in those cells undergoing a Ca(2+)-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca(2+) signal to activate Ca(2+)-dependent K(+) channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca(2+)-dependent K(+) channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca(2+) entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca(2+) entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4 alpha-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4 alpha-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca(2+) signal and the subsequent activation of Ca(2+)-dependent K(+) channels.