Related Experiment Video
Updated: Mar 26, 2026
Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents
Published on: July 3, 2013
Electrophysiological characterization of voltage-dependent calcium currents and TRPV4 currents in human pulmonary
Mozibur Rahman1, Subhendu Mukherjee1, Wei Sheng1
1Firestone Institute for Respiratory Health, St. Joseph's Hospital, Department of Medicine, McMaster University, Hamilton, Ontario, Canada; and.
Abstract:
We have presented indirect evidence of a key role for voltage-dependent Ca(2+) currents in TGFβ-induced synthetic function in human pulmonary fibroblast (HPF), as well as in bleomycin-induced pulmonary fibrosis in mice. Others, however, have provided indirect evidence for transient receptor potential vanilloid 4 (TRPV4) channels in both of those effects. Unfortunately, definitive electrophysiological descriptions of both currents in HPFs have been entirely lacking. In this study, we provide the first direct electrophysiological and pharmacological evidence of the currents in HPFs at rest and during overnight stimulation with TGFβ. These currents include a Ca(2+)-dependent K(+) current, a TRPV4 current, a chloride current, and an L-type voltage-dependent Ca(2+) current. Evidence for the TRPV4 current include activation of a large-conductance change by two putatively TRPV4-selective agonists (4α-phorbol-12,13-didecanoate; GSK1016790A), with a reversal potential near 0 mV, partial sensitivity to two different TRPV4-selective blockers (RN1734; HC067047), and partial reduction following removal of external Na(+) Substantial reduction of the evoked current was seen following the coapplication of RN1734, DIDS, and niflumic acid, suggesting that a chloride current is also involved. The voltage-dependent Ca(2+) current is found to be "L-type" in nature, as indicated by the voltage and time dependence of its activation, deactivation, and inactivation properties, and by its pharmacology (sensitivity to replacement with barium and inhibition by nifedipine, verapamil, or mibefradil). We also found that overnight treatment with TGFβ evoked a periodic current (inward at negative holding potentials, with reversal potential near 0 mV), which is sufficient to trigger the voltage-dependent Ca(2+) currents and, thereby, account for the rhythmic Ca(2+) oscillations, which we have described previously in these cells.
Insights
This study provides the first direct electrophysiological evidence of ion currents, including TRPV4 and L-type calcium currents, in human pulmonary fibroblasts. These findings clarify the role of these currents in TGFβ-induced fibrosis.
Area of Science:
- Cellular Electrophysiology
- Fibrosis Research
- Ion Channel Biology
Background:
- Indirect evidence suggests voltage-dependent Ca(2+) currents and TRPV4 channels play roles in TGFβ-induced fibroblast function and pulmonary fibrosis.
- Definitive electrophysiological characterization of these currents in human pulmonary fibroblasts (HPFs) has been lacking.
Purpose of the Study:
- To provide the first direct electrophysiological and pharmacological evidence of ion currents in HPFs at rest and during TGFβ stimulation.
- To characterize the nature of voltage-dependent Ca(2+) and TRPV4 currents in HPFs.
Main Methods:
- Electrophysiological recordings (voltage-clamp) were performed on HPFs.
- Pharmacological manipulation using selective agonists and blockers for TRPV4 channels and L-type Ca(2+) channels.
- Ionic substitution experiments (e.g., removal of external Na(+)) and assessment of current reversal potentials.
Main Results:
- Identified Ca(2+)-dependent K(+) current, TRPV4 current, chloride current, and L-type voltage-dependent Ca(2+) current in HPFs.
- TRPV4 current was activated by specific agonists, sensitive to blockers, and partially dependent on external Na(+).
- Voltage-dependent Ca(2+) current exhibited L-type characteristics (voltage/time dependence, pharmacology).
- TGFβ stimulation evoked a periodic current that can trigger L-type Ca(2+) currents, explaining previously observed Ca(2+) oscillations.
Conclusions:
- Direct electrophysiological evidence confirms the presence of multiple ion currents in HPFs, including TRPV4 and L-type Ca(2+) currents.
- These currents, particularly the TGFβ-evoked periodic current, are crucial for regulating Ca(2+) dynamics and potentially driving fibrotic processes.
- Findings provide a mechanistic basis for the role of ion channels in pulmonary fibrosis.
