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

Abstract

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 Ca(2+)-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 (P(o)) were -67, -45 and 85 mV with cytosolic free-Ca(2+) levels of 0.7 mM, 10 microM and 1 microM, respectively (n=8-14). The Hill coefficient for Ca(2+) 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 [Ca(2+)]. 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 microM 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 SLFs 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.

Deletion of the Ca2+-activated potassium (BK) α-subunit but not the BKβ1-subunit leads to progressive hearing loss

“…BK channels were shown to be present in the stria vascularis by immunocytochemistry (8) and electrophysiology (28) as well as in fibrocytes of the spiral ligament by electrophysiology (29). Although we cannot exclude effects on the endocochlear potential in BK␣ Ϫ/Ϫ mice older than those studied here, we were unable to detect any phenotypical change in the stria vascularis in BK␣ Ϫ/Ϫ mice, in line with the described profile of hearing loss.…”

Identification of ClC-2 and CIC-K2 Chloride Channels in Cultured Rat Type IV Spiral Ligament Fibrocytes

“…The amorphous material observed in the intercellular spaces in the cultures shared similar ultrastructural features with that seen in type I fibrocyte cultures and may represent collagen and fibronectin as in other fibroblast cell cultures (Goldberg and Green 1964;Hedman et al 1978;Gratton et al 1996). Also similar to type I fibrocytes in culture, the cultured type IV fibrocytes failed to form gap junction connections with one another even at confluence (Liang et al 2003). These results suggest that a particular microenvironment may be required to facilitate the formation of the extensive gap junctions that occur between spiral ligament fibrocytes in vivo.…”

“…The identity of the cultured cells was verified immunocytochemically with protein markers specific for various spiral ligament cell types. The cultured type IV fibrocytes appeared to maintain their in vivo immunostaining profile as do cultured type I fibrocytes (Gratton et al 1996;Liang et al 2003). The type IV fibrocytes appeared to have a higher cytoplasmic to nuclear area ratio and more abundant mitochondria than was seen in type I secondary cultures and also contained numerous small round vesicles beneath their plasma membrane.…”

“…The others were transferred to 35 Â 10-mm petri dish (Corning Inc., Corning, NY, USA) for primary cell culture. The explants were kept in a CO 2 incubator at 37-C and fed with media twice a week (Gratton et al 1996;Liang et al 2003).…”

“…It is probable that ClC-Ks mediate the exit of Cl _ imported by Na,K/ATPase and Na,K,Cl-cotransporter activities and maintain normal cellular electrical and ionic homeostasis in these fibrocytes (Crouch et al 1997;Qu et al 2006). Secondary cultures of type I spiral ligament fibrocytes have been successfully established (Gratton et al 1996;Suko et al 2000;Liang et al 2003;Shen et al 2004). Although these cells retain many of their in vivo characteristics and provide a good source for physiological studies, reports of the successful culture of other spiral ligament fibrocyte subtypes are lacking.…”