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A functional channel in kidney epithelial cells exhibit characteristics of gentamicin uptake‐related TRPV4 channel
Author(s) -
Jiang ZhiGen,
Wang Tian,
Karasawa Takatoshi,
Yang Yuqin
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.860.12
Subject(s) - chemistry , biophysics , reversal potential , trpv4 , patch clamp , channel blocker , ruthenium red , membrane potential , extracellular , microbiology and biotechnology , biochemistry , ion channel , biology , calcium , receptor , organic chemistry
Aminoglycosides (AGs) are important antibiotics but toxic to cochlear hair cells. Using kidney epithelial cell‐line cultures, we found that fluorescent gentamicin (GTTR) can enters MDCK and KPT2 cells independently of endocytosis and the uptake is enhanced by low [Ca 2+ ] o and by exogenous expression of TRPV4 in KPT2 cells (KPT2‐TRPV4 cells), suggesting an important role for TRPV4 in gentamicin transmembrane transport. In this study, using whole‐cell and patch‐clamp recording techniques, we identified a channel in MDCK and KPT2‐TRPV4 cells that exhibited characteristics of TRPV4: (1) In physiological pipette and bath solutions, cell‐attached and outside‐out patches from MDCK and KPT2‐TRPV4 cells frequently displayed unitary current activity with a reversal potential near zero mV and a single‐channel conductance of ~100 pS for outward current and 30 – 60 pS for inward current. The unitary activity (P o ) was enhanced by low extracellular Ca 2+ and suppressed by Gd 3+ , La 3+ and Ruthenium Red. (2) Hypotonic media (225 mOsmol), 100 μM ATP and 1 μM 4α‐phorbol 12,13‐didecanoate (4αPDD), a selective TRPV4 activator, stimulated this unitary activity. (4) The spontaneous or 4αPDD‐elicited unitary current was not sensitive to chloride channel blocker niflumic acid, flufenamic acid (FFA) or N‐phenylanthranilic acid. (3) In whole‐cell recordings, 4αPDD caused a net current with an I/V curve reversing its polarity near zero mV. (5) Control cells with empty vector (KPT2‐pBabe) showed very rare spontaneous NSC unitary currents. 4αPDD (2 μM) failed to elicit the unitary activity or whole‐cell I/V change in the KPT2‐pBabe cells. We conclude that MDCK and KPT2‐TRPV4 cells express functional TRPV4 channels; these data further substantiates the role of TRPV4 as a route for cellular gentamicin uptake and/or clearance. Funded by NIDCD R01 04716 (ZGJ), R03 DC009501 (T.K.)