A voltage- and Ca2+-dependent big conductance K channel in cochlear spiral ligament fibrocytes

Evidence is accruing that spiral ligament fibrocytes (SLFs) play an important role in cochlear K+ homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca2+-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298+/-5.6 pS (n=28) was measured under symmetrical K+. Membrane potentials for half-maximal open probability (PO) were -67, -45 and 85 mV with cytosolic free-Ca2+ levels of 0.7 mM, 10 muM and 1 muM, respectively (n=814). The Hill coefficient for Ca2+ affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P-o=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca2+]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086+/-0.021 mM and 0.99, respectively (n=4-9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7+/-4.5% (n=6) by 0.1 muM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (tau(o1) 0.23+/-0.08 MS, tau(o2) 1.40+/-0.32 ms; tau(c1) 0.26 +/- 0.09 ms, tau(c2) 3.10+/-1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel alpha subunit in cultured type I SLI's and freshly isolated spiral ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K+ recycling dynamics in the mammalian cochlea.