BK channels mediate the voltage-dependent outward current in type I spiral ligament fibrocytes

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BK channels mediate the voltage-dependent outward current in type I spiral ligament ¢brocytes Zhijun Shen

a;


, Fenghe Liang a , Debra J. Hazen-Martin a , Bradley A. Schulte

a;b

a

b

Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 165 Ashley Avenue, P.O. Box 250908, Charleston, SC 29425, USA Department of Otolaryngology-Head and Neck Surgery, Medical University of South Carolina, 165 Ashley Avenue, P.O. Box 250908, Charleston, SC 29425, USA Received 9 April 2003; accepted 14 October 2003

Abstract Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of Kþ recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I spiral ligament fibrocytes (SLFs), a major component of the Kþ recycling pathway, have a dominant Kþ membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70 6 1.2 mV at 1 nM intracellular free Ca2þ and shifted to 38 6 0.2 mV at 20 WM intracellular free Ca2þ (n = 4^6). The reversal potential of whole-cell tail currents against different bath Kþ concentrations was 52 mV per decade (n = 3^6). The sequence of relative ion permeability of the whole-cell conductance was Kþ s Rbþ ECsþ s Naþ (n = 5^17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC50 values of 0.07 mM and 0.013 WM, respectively (n = 3^7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly Kþ -selective and sensitive to IbTx (n = 4^9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be V6 s with repetitive voltage pulses from 370 to 80 mV (n = 3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the Kþ movement from perilymph to the stria vascularis. @ 2003 Published by Elsevier B.V. Key words: BK channel; Inactivation; K-recycling; Fibrocyte; Spiral ligament; Gerbil

1. Introduction Both the large Kþ gradient between endolymph and perilymph (normally V150/5 mM) and highly positive dc endocochlear potential (EP, V80 mV) are critical to mechanoelectrical transduction in the mammalian cochlea. While Kþ carries most of the current through inner ear sensory hair cells, the EP provides the essen* Corresponding author. Tel.: +1 (843)792-6454; Fax: +1 (843)792-2747. E-mail address: [email protected] (Z. Shen). Abbreviations: Cx, connexin; EGF, epidermal growth factor; EP, endocochlear potential; IbTx, iberiotoxin; SLFs, spiral ligament ¢brocytes; TEA, tetraethylammonium

tial driving force for acoustic transduction (Corey and Hudspeth, 1979; Kross et al., 1992; Zidanic and Brownell, 1990). The stria vascularis is considered the primary tissue site responsible for producing the high endolymphatic [Kþ ] and generating the positive EP (Takeuchi and Ando, 1999; Wangemann, 2002). However, evidence is rapidly accumulating that non-sensory cells, including highly specialized subpopulations of the spiral ligament ¢brocytes (SLFs), are active participants in these processes (Spicer and Schulte, 1996; Minowa et al., 1999; Steel, 1999). Maintaining a normal ionic gradient and high EP in the cochlea requires a dynamic and uninterrupted £ow of Kþ to the stria vascularis. Previous experiments have demonstrated that despite having a rich capillary net-

0378-5955 / 03 / $ ^ see front matter @ 2003 Published by Elsevier B.V. doi:10.1016/S0378-5955(03)00345-9

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work, the stria vascularis derives its Kþ supply mainly from perilymph (Konishi et al., 1978; Sterkers et al., 1982). This and other observations have led to the rapidly evolving concept of Kþ recycling (Schulte and Steel, 1994; Kikuchi et al., 1995, 2000; Spicer and Schulte, 1996, 1998; Steel, 1999; Wangemann, 2002). The principal idea is that cochlear Kþ gradients and the EP are maintained in part by sequential cellular Kþ transport activities that together form dynamic Kþ recycling pathways. Although both lateral and medial Kþ recycling pathways have been conceptualized (Spicer and Schulte, 1996, 1998), the former has received much more attention than the latter. Lateral Kþ recycling begins with Kþ entering outer hair cells from endolymph via apical mechanosensitive cation channels and exiting the cells to perilymph via basolateral voltage-dependent Kþ channels (Corey and Hudspeth, 1979; Santos-Sacchi et al., 1997). Much of this discharged Kþ is thought to be taken up by supporting cells in the organ of Corti, mainly tectal and Deiters’ cells, via the K^Cl cotransporter KCC4 (Boettger et al., 2002). The reabsorbed Kþ is presumed to move down to its electrochemical gradient through a gap junctioncoupled syncytium of cochlear supporting cells to the outer sulcus epithelium where it is released into spiral ligament perilymph (Kikuchi et al., 1995; Zhao and Santos-Sacchi, 2000). The released Kþ is then taken up by nearby type II SLFs through the combined activities of Na,K-ATPase and NKCC1. Theoretically, this resorbed Kþ £ows down its electrochemical gradient sequentially through type II and type I SLFs and strial basal and intermediate cells that together form a second gap junction-coupled syncytium in the lateral wall (Schulte and Adams, 1989; Crouch et al., 1997; Kikuchi et al., 1995; Sakaguchi et al., 1998). Kþ is released from intermediate cells into the intrastrial space, at least in part, through Kir4.1 (KCNJ10) channels that also serve as key elements in generation of the EP (Ando and Takeuchi, 1999; Takeuchi et al., 2000; Marcus et al., 2002). The recycling circuit is completed when Kþ transported into strial marginal cells via the combined e¡orts of basolateral Na,K-ATPase and NKCC1 is secreted back into endolymph via apical KCNQ1/ KCNE1 channels (Wangemann et al., 1995; Shen et al., 1997). In addition, Kþ di¡using into the scala tympani across the basilar membrane or the scala vestibuli across Reissner’s membrane provides another source of Kþ to feed the lateral wall syncytium via its active uptake by type IV and V SLFs. The precise molecular and cellular mechanisms governing Kþ recycling, especially its transit through the gap junction-coupled syncytia, remain poorly understood. Presumably, Kþ moves freely through gap junctions following its electrochemical gradient inside the syncytium. If true, the membrane potential and [Kþ ]

in the several di¡erent cell types forming the syncytium would determine the electrochemical gradient and thus drive Kþ recycling dynamics. Recently, we have identi¢ed and characterized a BK channel in cultured type I SLFs with the patch-clamp technique and con¢rmed the expression of BK channels in cultured and freshly isolated type I SLFs by reverse transcription polymerase chain reaction (Liang et al., 2003). We postulated that this channel signi¢cantly in£uences the cell membrane properties (Shen et al., 2001; Liang et al., 2002, 2003). Here we demonstrate that the BK channel is the dominant force responsible for regulating membrane conductance in type I SLFs, suggesting its critical role in establishing a proper electrochemical gradient for moving Kþ from SLFs into strial cells. These results were presented in part at recent meetings (Shen et al., 2001, 2002).

2. Materials and methods 2.1. Cell culture Fibrocyte cultures were derived from primary explants of the spiral ligament and the cells were characterized as type I ¢brocytes, as described previously (Gratton et al., 1996; Liang et al., 2003). In brief, 6^8 week old gerbils of either gender were anesthetized with pentobarbital (75^100 mg/kg, i.p.) and decapitated. The temporal bones were removed and the spiral ligament was rapidly dissected at 4‡C and minced into small fragments. These were transferred to 15U60 mm Petri dishes (one spiral ligament/dish) and placed in a CO2 incubator at 37‡C with maximal humidity and fed twice a week with the same medium. The cells were subcultured upon reaching con£uence and split every 7^10 days. The growth medium consisted of K-MEM supplemented with hydrocortisone (36 ng/ml), insulin (5 Wg/ ml), selenium (5 ng/ml), transferrin (5 Wg/ml), triiodothyronine (4 pg/ml), epidermal growth factor (EGF, 10 Wg/ml), 0.1% penicillin^streptomycin and 10% fetal bovine serum, all purchased from Gibco (St. Louis, MO, USA). 2.2. Whole-cell patch-clamp recording Fibrocytes from passages 3^5 were rinsed with sterile phosphate-bu¡ered saline and treated with 0.05% trypsin/EGTA for 3^5 min at room temperature. Enzyme activity was halted by adding serum-containing medium. The cells were collected in a 15 ml centrifuge tube and kept in a CO2 incubator for up to 8 h prior to transfer to a 40 Wl recording chamber mounted on an inverted microscope. The recording chamber was superfused at 0.5 ml/min with various bath solutions (see

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below). Recording pipettes were manufactured from Corning 7052 glass capillary using a programmable horizontal puller (Sutter, P-97, Novato, CA, USA) and heat polished to form a tip opening of around 1 Wm inside diameter. The recording pipette and reference electrode were connected to the headstage (CV203) of an Axonpatch 200B ampli¢er (Axon Instruments, Foster City, CA, USA) with Ag/AgCl wire. High resistance seals of 4^6 G6 were achieved regularly and conventional whole-cell con¢guration was established just after the formation of a high resistance seal. The average access resistance and membrane capacitance were 8.1 6 5.2 M6 and 11.7 6 5.0 pF (n = 16). Data were collected via an Axon 200B patch clamp ampli¢er and Axon Digidata 1200 A/D converter integrated with a Pentium II PC using an 80^85% compensation rate for all recordings. Signals were ¢ltered at 1 kHz by a built-in low-pass four-pole Bessel ¢lter and digitized at 5 kHz using a Digidata 1200 A/D converter (Axon Instruments). Microcal Origin (5.0) and pClamp (8.0) software were used for data collection and analysis. All recordings were done at room temperature. Speci¢c data analysis procedures are described in Section 3 and the ¢gure legends. Data are expressed as the mean 6 S.E.M. (n = number of cells). Statistical analysis was performed using Student’s t-test and a level of P 6 0.05 was considered signi¢cant. 2.3. Solutions and chemicals The pipette solution contained (in mM): 150 KCl, 1 Mg-ATP, 0.4 CaCl2 , 1 EGTA, 10 HEPES (pH 7.2). The free Ca2þ concentration of pipette solution was calculated to be 20 WM by a computer program (Schoenmakers et al., 1992). The bath solution contained (in mM): 145 NaCl, 4.5 KCl, 1 MgCl2 , 0.7 CaCl2 , 10 HEPES (pH 7.2). For testing univalent cations and tetraethylammonium (TEA)-containing solutions, NaCl was replaced by an equal amount of testing chemicals. Iberiotoxin (IbTx) and all other chemicals were purchased from Sigma (St. Louis, MO, USA). 2.4. Animal welfare A total of 10 gerbils were used for the study. Animal use and handling were approved by the Institutional Animal Care and Use Committee (IACUC) of the Medical University of South Carolina.

3. Results 3.1. Voltage and Ca2+ dependence With a 150 mM KCl pipette solution and a 145 mM

Fig. 1. The whole-cell current amplitudes (B and C) were highly dependent on the holding voltage levels of the testing protocol (A).

NaCl and 5 mM KCl bath solution, an outwardly rectifying whole-cell current was readily induced by depolarizing membrane potentials with a holding potential of 370 mV (Fig. 1C). The amplitude of the outward whole-cell current was reduced sharply when the holding potential was increased from 370 to 0 mV (Fig. 1B). The averaged half-maximal voltage activation value was obtained by ¢tting a Boltzmann function to the I^V plot (Fig. 3C). A slow and incomplete voltage-dependent inactivation was observed when longer episode voltage protocols were used (Figs. 2A and 3B). The recovery time was estimated to be V6 s using a protocol with a ¢xed holding voltage and variable durations (Fig. 2C, n = 3). The whole-cell current was highly dependent on the intracellular [Ca2þ ]. The current was robust in the presence of 20 WM [Ca2þ ]i , but was attenuated when 1 nM [Ca2þ ]i was present (Figs. 3A, B). This was best viewed by comparing voltage activation curves obtained under di¡erent experimental [Ca2þ ]i (Fig. 3C). The averaged half-maximal voltage activation potential was shifted by 32 mV, from 70 6 1.2 mV to 38 6 0.2 mV when [Ca2þ ]i was elevated from 1 nM to 20 WM (n = 4^6).

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tials (Fig. 4C, n = 3^6). This value is nearly ideal for a Kþ -permeable membrane conductance (Hille, 1991). The relative permeability of the whole-cell membrane conductance to univalent cations was evaluated by measuring reversal potentials with di¡erent testing cations in the bath. Assuming that Kþ was the only permeant ion inside the ¢brocyte and the testing cation was the only permeant ion outside the cell, the ratio of ion permeability is related to the reversal voltage by the Goldman^Hodgkin^Katz voltage equation: V r ¼ 3ðRT=F Þ lnðPk ½Ki =Px ½Xo Þ

ð1Þ

Fig. 2. Voltage-dependent inactivation of the whole-cell currents became evident when longer voltage pulses were applied (A,B). An interval of 6 s was required for a nearly total recovery when the current was elicited by repetitive pulses from 370 to 80 mV.

3.2. Ion selectivity The ion selectivity of the whole-cell current was investigated by ion substitution experiments. Reversal voltages of the tail current were measured against different concentrations of bath Kþ (Figs. 4A, B) and a slope of 52 mV per decade was obtained by ¢tting a linear regression function to the plot of reversal poten-

Fig. 3. Intracellular free Ca2þ levels signi¢cantly a¡ected both amplitudes and gating thresholds of the whole-cell currents (A^C).

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where R is the common gas constant, T the temperature and F the Faraday constant, Pk /Px is the ratio of the permeability of test cation X to Kþ . The average reversal voltages were 30.1 6 2.5 mV for Kþ , 310.5 6 2.2 mV for Rbþ , 354.2 6 4.8 mV for Csþ and 383.6 6 10.5 mV for Naþ (Fig. 4D, n = 5^17). The sequence of relative permeability of the whole-cell conductance was Kþ s Rbþ ECsþ s Naþ which is consistent with that of most known K channels, including the BK channel (Schlatter et al., 1993). 3.3. Pharmacological pro¢le The e¡ect of the non-speci¢c K channel blocker TEA was evaluated with bath application. TEA has been shown to reversibly block most K channels by binding to the Kþ -permeable pore (Hille, 1991). A dose^response curve of the whole-cell current to bath TEA was obtained with an IC50 of 0.07 6 0.05 mM (Figs. 5A, B, n = 3^6). The inhibitory e¡ect was partially reversible (data not shown). To more speci¢cally characterize the channel, a potent BK channel blocker IbTx was tested by bath ap-

Fig. 4. Tail currents were elicited by a testing protocol (A,B) and used to determine the reversal potentials for experimental conditions (C,D).

Fig. 5. Whole-cell currents were inhibited by extracellular TEA (A) and a dose^response curve was obtained (B).

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3.4. In vitro membrane potential (Em ) measurements Em was continuously measured in conventional whole-cell current clamp con¢gurations. With a 150 mM KCl pipette solution and a 145 mM NaCl/4.5 mM KCl bath solution, the averaged initial value of Em at break-in was 347.1 6 1.7 mV (n = 111). The averaged whole-cell membrane capacitance (Cm ) was 13.5 6 2.2 pF (n = 98). The Em gradually shifted to an average of 380.6 6 1.2 mV within 3 min and became stable thereafter (n = 9). The new Em level was near the equilibrium potential for a dominant K membrane conductance, which was further con¢rmed by ion substitution experiments. The Em shifted to 0 mV when the 145 mM NaCl bath solution was replaced by a 150 mM KCl solution and returned to near 380 mV upon switching back to the control bath solution (Fig. 7, n = 9). The K-selective Em indicated the possible involvement of the BK channel, which was tested by application of IbTx. The Em was highly sensitive to a bath solution containing 0.1 WM IbTx (Fig. 8, n = 4).

4. Discussion

Fig. 6. A highly sensitive e¡ect of IbTx can be seen in A and a dose^response curve is displayed in B.

plication. The whole-cell outward current was highly sensitive to IbTx, which did not require constant membrane depolarization (Fig. 6A). The normalized inhibitory e¡ect of IbTx on whole-cell current was plotted against three concentrations of IbTx. An IC50 value of 0.013 WM was determined by ¢tting a Hill function to the dose^response curve (Fig. 6B, n = 3^7). The inhibitory e¡ect of IbTx on the whole-cell current was totally reversible shortly after control bath solution was reintroduced (data not shown).

The concept of Kþ recycling in the inner ear is rapidly gaining credence based on results from both basic and clinical research studies. Histological as well as functional changes consistent with alterations in Kþ homeostasis have been associated with genetic mutations in elements of the proposed Kþ recycling pathway including the KCC4 and NKCC1 cotransporters, the KCNQ4 K, KCNJ10, KCNQ1 and KCNE1 K channel subunits, and the gap junction connexins Cx26, Cx30, Cx31, Cx32 and Cx43 (Boettger et al., 2002; Rabionet et al., 2000; Delpire et al., 1999; Minowa et al., 1999; Dixon et al., 1999; Marcus et al., 2002; Vetter et al., 1996). Some of these mutations have now been linked to previously identi¢ed forms of non-syndromic hearing loss (Kubisch et al., 1999; Janecke et al., 2000; Liu et al., 2000, 2001; Wang et al., 2002; Tyson et al., 1997).

Fig. 7. An example of in vitro Em measurement is shown. The responses of Em to changes of extracellular [Kþ ] indicate that the membrane potential is primarily controlled by K conductance.

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Fig. 8. The role of BK channels in the K-selective Em was evidenced by signi¢cant inhibition of the Em with 1.0 WM of IbTx (A,B).

In addition, a recent study employing perfusion of a gap junction-decoupling agent into the round window membrane niche via a mini osmotic pump has documented signi¢cant changes in cochlear histology and function consistent with the Kþ recycling theory (Spiess et al., 2002). Localized beneath the stria vascularis, type I SLFs occupy a central position in the lateral wall syncytium, making contact with one another and with type II, IV and V SLFs and strial basal cells via gap junctions. The ¢nding here that BK channels dominate the voltage-dependent membrane conductance in type I SLFs suggests that they may play a major role in generating and regulating the electrochemical gradient inside the lateral wall syncytium, a process crucial to Kþ recycling (Schulte and Steel, 1994; Kikuchi et al., 1995, 2000; Spicer and Schulte, 1996, 1998; Steel, 1999; Wangemann, 2002). Indeed, dominant Kþ membrane conductance has been reported in all cell types thus far studied in the lateral wall syncytium (Liang et al., 2001, 2002; Takeuchi and Ando, 1999; Takeuchi and Irimajiri, 1996; Takeuchi et al., 2000). These ¢ndings are consistent with the need to generate a membrane potential gradient facilitating the hypothetical movement of Kþ through SLFs to the stria vascularis because the extracellular [Kþ ] in perilymph surrounding SLFs (V5 mM) is only slightly higher than that in the intrastrial £uid bathing strial basal and intermediate cells (V1.5^3 mM) (Salt et al., 1987; Ikeda and Morizono, 1989). The BK channels in type I SLFs exhibit some novel properties previously only observed in excitable cells. The half-maximal membrane activation potential was calculated to be 38 6 0.2 mV with a 20 WM free intra-

cellular Ca2þ level. Given that the membrane potential of SLFs has been estimated to be 0 to 310 mV in vivo, the BK channels theoretically would be closed under physiological conditions, unless other regulatory mechanisms are operative (Salt et al., 1987; Ikeda and Morizono, 1989; Melichar and Syka, 1987). The voltage dependence of BK channels generally shifts towards hyperpolarization with increasing intracellular free Ca2þ levels. We recently reported the presence of a voltage-dependent L-type Ca channel in type I SLFs with a maximal membrane activation potential near 0 mV (Hu et al., 2003). Under physiological conditions, this channel would remain open and promote Ca2þ in£ux, since free Ca2þ levels in perilymph are much higher than those in cytosol (Smith et al., 1954). Presumably, this Ca channel works together with an intracellular Ca-ATPase (Schulte, 1993) to regulate cytosolic Ca2þ levels and BK channel activity in type I SLFs. Another recent study has demonstrated that the activity of BK channels in type I SLFs is directly regulated by dephosphorylation (Liang et al., 2002). Dephosphorylation increased the open probability in single-channel recordings, increased whole-cell current amplitudes and shifted the voltage dependence curve towards a hyperpolarized state. Together, these ¢ndings argue favorably for the active participation of BK channels in maintaining the membrane potential of type I SLFs under physiological conditions. A second biophysical feature of BK channels in type I SLFs is their voltage-dependent incomplete inactivation. Voltage-dependent BK channel inactivation is commonly seen in neurons and is usually regulated by L subunit modulation. This regulatory activity imparts

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to neurons biophysical properties similar to those governed by K inward recti¢ers in cardiac myocytes, allowing the cells to maintain their membrane potential while conserving intracellular Kþ (McManus et al., 1995; Wallner et al., 1995; Wallner et al., 1999; Brenner et al., 2000). Such regulatory mechanisms would appear to be critical to the function of type I SLFs, since these cells are thought to transmit Kþ intracellularly through the gap junction-coupled spiral ligament ¢brocyte network for return to the stria vascularis (Schulte and Steel, 1994; Spicer and Schulte, 1996). In summary, our data demonstrate that type I SLFs exhibit a dominant BK channel conductance that likely contributes to establishing and maintaining an electrochemical gradient essential for Kþ recycling.

Acknowledgements This work was supported by Grants DC5148 and DC00713 from the NIDCD/NIH.

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