ABSTRACT The large conductance Ca2+-activated K+ channel, BK (KCNMA1), is expressed along the connecting tubule (CNT) and cortical collecting duct (CCD) where it underlies flow- and Ca2+-dependent K+ secretion. Its activity is partially under the control of the mechanosensitive TRPV4 Ca2+-permeable channel. Recently we identified three small/intermediate conductance Ca2+-activated K+ channels, SK1 (KCNN1) and SK3 (KCNN3), and IK1 (KCNN4), with notably high Ca2+-binding affinities, that are expressed in CNT/CCD and may be regulated by TRPV4-mediated Ca2+ influx. The K+-secreting CCD mCCDcl1 cells, which express these channels, were used to determine if SK1/3 (SK1, SK3) and IK1 are activated upon TRPV4 stimulation and if they contribute to Ca2+ influx and activation of BK. Activation of TRPV4 (GSK1016790A) modestly depolarized the membrane potential and robustly increased intracellular Ca2+, [Ca2+]i. Inhibition of both SK1/3 and IK1 by application of apamin and TRAM-34, respectively, further depolarized the membrane potential and markedly suppressed the TRPV4-mediated rise in [Ca2+]i. Application of BK inhibitor, Iberiotoxin, after activation of TRPV4 without apamin/TRAM-34, also reduced [Ca2+]i and further intensified membrane depolarization, demonstrating BK involvement. However, the BK dependent effects on [Ca2+]i and membrane potential were largely abolished by pretreatment with apamin and TRAM-34, identical to that observed by separately suppressing TRPV4-mediated Ca2+ influx, demonstrating that SK1/3-IK1 channels potently contribute to TRPV4-mediated BK activation. Our data indicate a direct correlation between TRPV4-mediated Ca2+ signal and BK activation, but where early activation of SK1/3 and IK1 channels are critical to sufficiently enhanced Ca2+ entry and [Ca2+]i levels required for activation of BK.
- KCa Channels
- Cortical collecting duct
- mCCDcl1 cells
- intracellular Ca2+
- Copyright © 2017, American Journal of Physiology-Renal Physiology