The outer hair cell motor in membrane patches

 We have used patch-clamp techniques to record the charge movement associated with motility in patches of basolateral membrane from isolated outer hair cells. Charge movement has been measured from the voltage-dependent capacitance. Using 3 to 4 MΩ pipettes with tip diameters of 3 μm the measured maximum voltage-dependent capacitance was 56 ± 6 fF at –36 mV when the resting membrane potential was –20 mV. The calculated total charge movement was 5.6 ± 0.6 fC (n = 13) and the inferred density of univalent motor elements was 8400/μm². Negative pressure (applied via the pipette) increased membrane tension and shifted the capacitance peak to depolarised potentials. Under conditions of isotropic membrane stress there was no change in the peak measured capacitance in contrast to that measured in previous whole-cell recordings. Received: 19 December 1996 / Received after revision: 21 February 1997 / Accepted: 5 March 1997     

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca²⁺-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3±0.3 pS (n=15) under symmetrical inger conditions. The channel excluded anions (P _Na/P _Cl=18 whereP _Na/P _Cl denotes the relative permeability of Na to Cl); it was equally permeant to the Na⁺ and K⁺ ions and exhibited a low permeability toN-methyl-D-glucamine and Ba²⁺ or Ca²⁺. Channel opening required a free Ca²⁺ concentration of about 10^–6 mol/l on the internal side of the membrane and the open probability (P _o) was maximal at 10^–3 mol/l (P _o=0.72±0.06,n=12). Adenosine 5mono-, tri- and di-phosphate reducedP _o to 29±14 (n=5), 42±10 (n=8) and 51±12 (n=5) % of controlP _o, respectively, when they were added at a concentration of 10^–3 mol/l to the internal side. The channel was partially blocked by flufenamic acid (10^–4 mol/l) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10^–5 mol/l). This type of channel, together with Ca²⁺-activated K⁺ channels, might participate in the control of membrane potential and modulate the motility of OHCs.     

The effects of changing intracellular pH on calcium and potassium currents in smooth muscle cells from the guinea-pig ureter

 Guinea-pig ureteric smooth muscle is unusual in that intracellular acidification increases and alkalinization decreases force production. To help elucidate the mechanism underlying these effects on force we have investigated the effects of changing intracellular pH on both calcium and potassium currents in single cells isolated from the guinea-pig ureter to determine their possible role in force development. Depolarization to +40 mV resulted in a fast transient outward current which was inhibited by 4-aminopyridine but not tetraethylammonium. Intracellular alkalinization (20 mM trimethylamine) increased this current to 179 ± 24% of the control and resulted in the development of a slowly activating large outward current which was inhibited by tetraethylammonium and washout. Acidification (40 mM sodium butyrate) decreased the fast transient outward current to 58 ± 3% of the control and did not produce a slowly activating current. When potassium was replaced by caesium in the pipette solution, depolarization to 0 mV resulted in an inward calcium current which was abolished by nifedipine. Intracellular alkalinization increased this current to 126 ± 11% of the control whereas acidification had the opposite effect, decreasing it to 55 ± 10%. Furthermore, current-clamp experiments showed that intracellular alkalinization inhibited the amplitude of the action potential, therefore decreasing excitability of the cell. From our results, we suggest that the predominant effects of intracellular pH on force production in the guinea-pig ureter are mediated via the modulation of outward potassium currents (thereby reducing excitability of the tissue) rather than the effects on the inward calcium current. Received: 31 July 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Sound-evoked efflux of excitatory amino acids in the guinea-pig cochlea in vitro

We have used the perfused guinea-pig temporal-bone preparation to study the sound-evoked efflux of aspartate and glutamate, which are putative afferent transmitters in the cochlea. The cochlea was stimulated with white noise at 89, 95, and 101 dB SPL. Cochlear function was monitored by recording the endocochlear potential, the cochlear microphonic, and the summating potential. In silence, there was a low basal efflux of both amino acids. A significant and intensity dependent sound-evoked efflux of aspartate was observed at all levels, whereas a significant efflux of glutamate was found only at the 101 dB SPL level. Immunohistochemistry of sections from the organ of corti showed an ubiquitous distribution of glutamate-like immunoreactivity in the sensory organ and ganglion, whereas aspartate-like immunoreactivity was found in the region of the inner hair cells and in the spiral ganglion. In view of these findings, we suggest that not only glutamate, but also aspartate may have a neurotransmitter role in the afferent pathway of the cochlea. Received: 7 November 1997 / Accepted: 3 March 1998     

Ionic currents and current-clamp depolarisations of type I and type II hair cells from the developing rat utricle

 Ionic currents and the voltage response to injected currents were studied in an acutely dissected preparation of the rat utricle between birth and postnatal day 12 (PN12). Based upon morphological criteria, the sensory cells examined were divided into two classes, ”type I” and ”type 2 category,” the latter of which may include some immature type I cells. The former group comprises a clearly defined electrophysiological population, with one large outwardly rectifying potassium conductance that is sensitive to 4-aminopyridine (4-AP), insensitive to tetraethylammonium (TEA) and displays voltage-dependent activation kinetics. In the absence of enzymatic dissociation procedures, and with the epithelium left largely intact, the mean half activation of this conductance was –30.3 mV at PN3, and –37.5 mV at PN12. At both stages it was almost entirely turned off at –74 mV. Omission of ATP from the intracellular solution appeared to prevent rundown of this conductance. Type II category hair cells formed a more heterogeneous population, exhibiting a distinct TEA-sensitive delayed rectifier potassium conductance; the rapidly activating and inactivating I _A; an inward rectifier; and inward sodium currents at around PN3. Both cell types depolarised strongly in response to injected currents, with time courses reflecting the activation kinetics of their major outward conductances. Received: 11 July 1998 / Received after revision: 19 January 1999 / Accepted: 16 February 1999     

Endolymphatic and intracellular resting potential in the alligator lizard cochlea

Summary Endolymphatic potential (EP) and intracellular resting potentials (RPs) in the cochlea of the alligator lizard were measured with micropipets. The EP (defined as the dc electric potential recorded between the endolymph in scala media and the perilymph in scala tympani or scala vestibuli) had a mean value of +16 mV (in 96 ears). The RPs (defined as the de potentials recorded between the intracellular and perilymphatic spaces) were different in the distal portion of the cochlear nerve (range to-56 mV, mean of –23 mV in 158 cells) from those in the region of the basilar papilla (range to-154 mV, mean of-73 mV in 623 cells). In identified cells in the region of the basilar papilla, the mean values of RPs of hyaline epithelial cells (-113 mV in 7 cells) and supporting cells (-93 mV in 13 cells) were more negative than of hair cells (-73 mV in 5 cells). Differences in measured RPs could reflect differences in the resting potentials of these cells and/or their source resistances. RPs measured in the basilar papilla of the alligator lizard are compared with those obtained in other vertebrate hair cell organs.     

Axial and transverse stiffness measures of cochlear outer hair cells suggest a common mechanical basis

 The function of the hearing organ is based on mechanical processes occurring at the cellular level. The mechanical properties of guinea-pig isolated sensory cells were investigated using two different techniques. The stiffness of the outer hair cells along the longitudinal axis was measured by compressing the cell body using stiffness-calibrated quartz fibres. For cells with a mean length of 69 μm, the mean axial compression stiffness was 1.1±0.8 mN/m (±SD). There was an inverse relation between stiffness and cell length. The stiffness of the cell membrane perpendicular to the longitudinal axis of the sensory cell was measured by indenting the cell membrane with a known force. The mean lateral indentation stiffness was 3.3±1.5 mN/m (±SD) for cells with a mean length of 64 μm. Longer cells were less stiff than short cells. Modelling the hair cell as a shell with bending resistance, finite element calculations demonstrated that the axial compression stiffness correlated well with the lateral indentation stiffness, and that a simple isotropic model is sufficient to explain the experimental observations despite the different stress strain states produced by the two techniques. The results imply that the two different stiffness properties may originate from the same cytoskeletal structures. It is suggested that the mechanical properties of the outer hair cells are designed to influence the sound-induced motion of the reticular lamina. In such a system, stiffness changes of the outer hair cell bodies could actively control the efficiency of the mechanical coupling between the basilar membrane and the important mechanoelectrical transduction sites at the surface of the hearing organ. Received: 12 June 1997/Received after revision: 13 November 1997/Accepted: 19 January 1998     

Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea

Cyclopiazonic acid (CPA) is a reticulum-like intracellular Ca(2+) store depletory, which raises intracellular Ca(2+) concentration. The effect of CPA on membrane currents in isolated inner hair cells (IHCs) from guinea-pig cochlea was investigated by the patch-clamp technique in the whole-cell configuration. Four out of eight IHCs showed an augmentation of the currents and the other four cells showed an inhibition of the currents by extracellular CPA application. The activation kinetics of outward currents were not changed by CPA. Three out of four IHCs obtained from the basal part of the cochlea demonstrated augmentation, whereas three out of four IHCs from the apical part demonstrated inhibition of the currents. This result suggests that Ca(2+)-activated currents were dominant in the basal IHCs of the cochlea.     

Patch clamped responses from outer hair cells in the intact adult organ of Corti

Outer hair cells (OHCs) from the mammalian cochlea act as both sensory cells and motor cells. We report here whole-cell tight seal recordings of OHC activity in their natural embedding tissue, the intact organ of Corti, using a temporal bone preparation. The mean cell resting potential, –76±4 mV (n=19) and input conductance (10±3 nS at –70 mV) of third turn hair cells were significantly lower than have been found in isolated cells. Two main K⁺ currents in the cell were identified. One current, activated positive to –100 mV, was reduced by 5 mM BaCl₂. The other current, activated above –40 mV, was reduced by 100 M 4-aminopyridine (4-AP) and by 30 mM tetraethylammonium (TEA). Both of these currents have been also identified in recordings reported from isolated cells. On stepping to different membrane potentials, cells imaged in the organ of Corti changed length by an amount large enough to cause visible distortions in neighbouring cells. By quantifying such distortions we estimate that the forces generated by OHCs can account for the enhanced response to sound required by the cochlear amplifier.     

Ionic currents expressed in a cell line derived from the organ of Corti of the Immortomouse

 We have investigated the maturation of adult hair cell electrophysiology in a population of precursor cells in a conditionally immortal cell line. The cell line, UB/OC-2, from the embryonic organ of Corti of the H-2Kb-tsA58 transgenic mouse, permits cells to grow proliferatively at 33°C and to differentiate at 39°C. Whole-cell patch-clamp recordings showed that proliferating cells had a different electrophysiology to differentiating cells. Differentiating cells had a conditionally expressed slowly activating inward current activated by hyperpolarization. The current was not blocked by extracellular application of 0.5 mM Ba²⁺, but was blocked reversibly by 2 mM Cs⁺. The current was found to be carried by both K⁺ and Na⁺ ions (P _K/P _Na=2.2) and activated by 10 μM forskolin. These properties identify the slowly activating current as I _h. A proportion of proliferating and differentiating cells exhibited a voltage-gated Na⁺ current, I _Na. I _Na was abolished in Na-free external medium and was inhibited reversibly by tetrodotoxin (TTX) with K _i=64 nM. Together these results suggest that proliferating and differentiating hair cell precursors in the immortal cochlear cell line UB/OC-2 express currents which are also found in developing hair cells. Received: 6 January 1999 / Received after revision: 1 February 1999 / Accepted: 2 February 1999     

Heterogeneity of hair cells in the bullfrog sacculus

 Morphometric and electrophysiological features of hair cells in the sacculus of the bullfrog (Rana catesbeiana) were studied. Confocal microscopy observations of hair cells in situ revealed that three classes of hair cells can be distinguished by their somata shape and macular location. Two of these, termed central cylindrical (CCHCs) and central flask-shaped (CFHCs) hair cells, were found in the central part of the macula. The third class, termed peripheral elongated hair cells (PEHC), was only found around the perimeter of the sacculus. Using the whole-cell patch-clamp technique CCHCs and CFHCs were distinguished by the amplitude of their voltage-activated calcium currents (I _Ca). The mean amplitudes of steady-state I _Ca at –20 mV were –900 ± 500 pA (n = 18) for CCHCs and –160 ± 70 pA (n = 10) for CFHCs. The two hair-cell types also differed in the possession of a Cs⁺-resistant, apamin-sensitive, calcium-sensitive potassium current, found only the CFHCs. This study indicates that several populations of hair cells with distinct morphological features exist in the bullfrog sacculus, and that at least two of these differ in their complement of membrane conductances. Received: 18 February 1997 / Received after revision: 24 June 1997 / Accepted: 23 July 1997     

Regulation of stably expressed and native BK channels from human myometrium by cGMP- and cAMP-dependent protein kinase

 The cloned BK channel α subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOα cells) or in combination with the auxiliary β subunit (CHOα+β cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of α and β subunits enhanced the Ca²⁺ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the β subunit. The cGMP kinase Iα and cAMP kinase failed to affect BK channel activity in CHOα and CHOα+β cells at different [Ca²⁺]_i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NP _o), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NP _o 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study. Received: 17 March 1998 / Received after revision: 15 May 1998 / Accepted: 28 May 1998     

Inwardly rectifying K+ currents in fetal alveolar type II cells: regulation by protein kinase A and protein phosphatases

Fetal guinea-pig lung alveolar type II (ATII) cells have inwardly rectifying (IR) K⁺ currents that display Mg²⁺- and G-protein-dependent run-down. We have used the whole-cell patch-clamp technique to investigate further the regulation of these currents. Under control conditions [KCl-rich pipette solution (1 mM free Mg²⁺, 10 nM free Ca²⁺) and KCl-rich bath solution], we found that IR K⁺ currents diminished with a t _1/2 of 7.6 min and were absent by 30 min. Experimental manoeuvres designed to inhibit phosphorylation increased the rate of current run-down. Thus, intracellular addition of 100 μM H-7, a general kinase inhibitor, reduced the t _1/2 to 4.7 min and the currents were absent by 16 min. Similarly, protein kinase A (PKA) inhibitor peptide (50 nM) also accelerated run-down. Agents known to increase phosphorylation, such as db-cAMP (0.5 mM) and forskolin (10 μM), resulted in a significant slowing of run-down (t _1/2>16 min) as did intracellular addition of the catalytic subunit of PKA (100 nM). Similarly, inhibition of dephosphorylation by either 1 μM okadaic acid [protein phosphatase 1/2A (PP-1/2A) inhibitor] or anti-human protein phosphatase 2Cα (PP2C) antiserum decreased the rate of run-down. These results indicate that the phosphorylation-dependent activation state of the fetal ATII cell IR K⁺ channel is regulated by a complex interplay of kinases and phosphatases involving PKA (activation), and PP2C and PP-1/2A (inactivation). Received: 10 February 1999 / Received after revision and accepted: 31 March 1999     

cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes

 The effect of guanosine 3′,5′-cyclic monophosphate (cGMP) on L-type Ca current (I _Ca) was investigated in a study of rabbit ventricular myocytes using the whole-cell patch-clamp technique. Intracellular application of cGMP (100 μM) increased I _Ca in the absence of isoprenaline or forskolin. 8-Bromo-cGMP (100 μM) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP, 400 μM), relatively specific stimulators of cGMP-dependent protein kinase (cGMP-PK), also increased I _Ca. The stimulatory effect of 8-pCPT-cGMP was suppressed by Rp-8-chlorophenylthio-cGMP (400 μM), a phosphodiesterase-resistant cGMP-PK inhibitor. When I _Ca was increased by bath application of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM), 8-pCPT-cGMP (400 μM) resulted in additional stimulation of I _Ca. In the presence of 8-pCPT-cGMP, additional applications of isoprenaline (1 μM) or forskolin (1 μM) induced a further increase in I _Ca. From these results, it could be concluded that the activation of cGMP-dependent protein kinase is involved in the facilitation of I _Ca by cGMP in rabbit ventricular myocytes. Received: 17 March 1997 / Received after revision: 28 August 1997 / Accepted: 16 September 1997     

Regulation of the voltage-gated potassium channel KCNQ4 in the auditory pathway

The potassium channel KCNQ4, expressed in the mammalian cochlea, has been associated tentatively with an outer hair cell (OHC) potassium current, I_K,n, a current distinguished by an activation curve shifted to exceptionally negative potentials. Using CHO cells as a mammalian expression system, we have examined the properties of KCNQ4 channels under different phosphorylation conditions. The expressed current showed the typical KCNQ4 voltage-dependence, with a voltage for half-maximal activation (V_1/2) of –25 mV, and was blocked almost completely by 200 µM linopirdine. Application of 8-bromo-cAMP or the catalytic sub-unit of PKA shifted V_1/2 by approximately –10 and –20 mV, respectively. Co-expression of KCNQ4 and prestin, the OHC motor protein, altered the voltage activation by a further –15 mV. Currents recorded with less than 1 nM Ca²⁺ in the pipette ran down slowly (12% over 5 min). Buffering the pipette Ca²⁺ to 100 nM increased the run-down rate sevenfold. Exogenous PKA in the pipette prevented the effect of elevated [Ca²⁺]_i on run-down. Inhibition of the calcium binding proteins calmodulin or calcineurin by W-7 or cyclosporin A, respectively, also prevented the calcium-dependent rapid run-down. We suggest that KCNQ4 phosphorylation via PKA and coupling to a complex that may include prestin can lead to the negative activation and the negative resting potential found in adult OHCs.     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle

 To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca²⁺] ([Ca²⁺]_i)and phasic force showed that Wortmannin (1–4 μM) abolishes force with little or no change in [Ca²⁺]_i and electrical activity. High-K⁺-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent – at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40–50%. It also significantly increased the delay between the Ca²⁺ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca²⁺ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca²⁺ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered. Received: 4 June 1998 / Recived after revision: 3 July 1998 / Accepted: 3 July 1998     

Denervation increases protein tyrosine kinase and protein tyrosine phosphatase activities in fast and slow skeletal muscle

We have investigated the expression and regulation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in fast extensor digitorum longus (EDL) and slow soleus (SOL) in adult rat skele-tal muscles. Biochemical assays revealed significantly greater PTK and PTP activities in SOL than in EDL; these results were confirmed and extended by in-gel assays demonstrating that the PTKs and PTPs detected had higher activity levels in SOL compared to EDL extracts. Although phosophotyrosine proteins were concentrated at the neuromuscular junction (NMJ), PTK and PTP activities were observed in extra-junctional regions of the muscle fiber. Following denervation, we observed significant increases in PTK and PTP activities in both SOL and EDL, and gel-based assays showed an increase in the activities of several PTKs and PTPs. These results suggest that the same PTK and PTPs have different activity levels in fast and slow skeletal muscles and are regulated by nerve-dependent mechanisms. Accepted: 31 May 2001