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

Abstract

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.

Effect of osmotic stress on the expression of TRPV4 and BKCa channels and possible interaction with ERK1/2 and p38 in cultured equine chondrocytes

“…The current study showed an increase in TRPV4 channel expression at the protein level following hypo-osmotic challenge. This finding was in agreement with other reports in bronchial endothelial cells (12) and porcine articular chondrocytes (33). Several hypotheses have been proposed to implicate the BK Ca channel in cell volume regulation.…”

“…Differentiating between these two hypotheses is rather difficult, as cell swelling is associated with membrane stretch. Previous studies reported a coupling between TRPV4 and BK Ca channels in the vascular smooth muscle response to vasodilatory factors through the ryanodine receptor (RYR) (7), whereas in bronchial endothelial cells the RYR is not involved in the direct coupling between the two channels in response to hypotonic stress (12).…”

Functional Apical Large Conductance, Ca2+-activated, and Voltage-dependent K+ Channels Are Required for Maintenance of Airway Surface Liquid Volume

“…This activation pattern may be important in the airway but remains unexplored here. In the human bronchial epithelial cell line 16HBE14, BK responds to hypotonic stress (60) and to high viscosity through TRPV4/BK interactions (61).…”

IP3 Receptor Binds to and Sensitizes TRPV4 Channel to Osmotic Stimuli via a Calmodulin-binding Site

“…TRPV4 participation in osmo-and mechanotransduction (1,2) contributes to important functions including cellular (3,4) and systemic volume homeostasis (5,6), arterial dilation (7,8), nociception (9), epithelial hydroelectrolyte transport (10,11), bladder voiding (12), and ciliary beat frequency regulation (13)(14)(15). TRPV4 also responds to temperature (16,17), endogenous arachidonic acid (AA) metabolites (18), and phorbol esters including the inactive 4␣-phorbol 12,13-didecanoate (4␣PDD) (19,20) and participates in receptor-operated Ca 2ϩ entry (15), thus showing multiple modes of activation.…”