Biomedical subjects

J F Ashmore

Publications and source records attributed to J F Ashmore.

At least 19 recordsLinked to original sources

PubMed2005

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 microM 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 Ca(2+) in the pipette ran down slowly (12% over 5 min). Buffering the pipette Ca(2+) to 100 nM increased the run-down rate sevenfold. Exogenous PKA in the pipette prevented the effect of elevated [Ca(2+)](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.

Animals↗

PubMed2004

Apical endocytosis in outer hair cells of the mammalian cochlea.

Outer hair cells (OHCs), the sensory-motor cells of the mammalian cochlea, contain an endocytic tubulovesicular compartment below their apical stereocilia. We have used two-photon imaging of FM1-43 in the intact epithelium to show that these cells take up membrane in a Ca(2+)-dependent manner from a distinct apical site. The uptake rate was 0.8 microm(2)/s and internalized membrane was trafficked rapidly to a compartment along the lateral wall and distinct intracellular compartments. Double labelling with FM1-43 and DiOC(6), an endoplasmic reticulum (ER) marker, showed that these compartments are part of the tubulovesicular endoplasmic reticulum of OHCs. Labelling with a lysosomal marker showed that OHC lysosomes are restricted to the apex. Using the protein marker wheat germ agglutinin (WGA-FITC) we demonstrate that apical protein internalization and trafficking is about eight times slower than membrane internalization. Using double labelling with FM1-43 and WGA-FITC, we show that membrane and protein internalization are apically colocalized but that patterns of protein and membrane traffic differ. Protein was targeted only to the most apical third of the lateral wall. In control conditions, OHCs displayed only weak WGA-FITC surface labelling at the site of endocytosis. Lowering the rate of apical endocytosis increased this surface signal. The results suggest that OHCs endocytose membrane and membrane proteins with a high turnover rate and that these cells may use apical endocytosis to sort proteins via an indirect pathway to the lateral membrane.

Animals↗

PubMed2003

Sugar transport by mammalian members of the SLC26 superfamily of anion-bicarbonate exchangers.

The mammalian cochlea contains a population of outer hair cells (OHCs) whose electromotility depends on an assembly of 'motor' molecules in the basolateral membrane of the cell. Named 'prestin', the molecule is a member of the SLC26 anion transporter superfamily. We show both directly and indirectly that SLC26A5, rat prestin, takes up hexoses when expressed in several cell lines. Direct measurements of labelled fructose transport into COS-7 cells expressing prestin are reported here. Indirect measurements, using imaging techniques, show that transfected HEK-293 or CHO-Ki cells undergo reversible volume changes when exposed to isosmotic glucose-fructose exchange. The observations are consistent with the sugar transport. A similar transport was observed using a C-terminal green fluorescent protein (GFP)-tagged pendrin (SLC26A4) construct. Cells transfected with GFP alone did not respond to sugars. The data are consistent with fructose being transported by prestin with an apparent Km=24 nm. From the voltage-dependent capacitance of transfected cells, we estimate that 250,000 prestin molecules were present and hence that the single transport rate is not more than 3000 fructose molecules s(-1). Comparison of the transfected cell swelling rates induced by fructose and by osmotic steps indicates that water was co-transported with sugar. We suggest that the structure of SLC26 family members allows them to act as neutral substrate transporters and may explain observed properties of cochlear hair cells.

Algorithms↗

PubMed2001

Purinergic control of intercellular communication between Hensen's cells of the guinea-pig cochlea.

1. Hensen's cells in the isolated cochlea were stimulated by extracellular adenosine 5'-triphosphate (ATP) applied to their endolymphatic surface while changes in membrane current and intracellular calcium concentration ([Ca2+]i) were measured simultaneously. The response consisted of (i) an initial rapid inward current accompanied by elevation of the [Ca2+]i, (ii) a more slowly rising inward current accompanied by a rise of the [Ca2+]i and (iii) a slowly developing reduction of input conductance. 2. The slower responses were maintained in the absence of extracellular Ca2+. Similar responses were produced by increasing the [Ca2+]i via UV flash photolysis of intracellular D-myo-inositol 1,4,5-trisphosphate, P4(5)-(1-(2-nitrophenyl)ethyl) ester (caged InsP3) loaded at pipette concentrations of 8-16 microM. 3. The slow inward current, reversing around 0 mV, was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). 4. Bath application of U-73122 (1 microM), a phospholipase C inhibitor, eliminated the slow Ca2+-release component of the response to ATP. It is proposed that the effects of ATP are mediated by the co-activation of ionotropic P2X and metabotropic P2Y receptors. 5. Immunohistochemistry using light and electron microscopy revealed that inositol 1,4,5-trisphosphate (InsP3) receptors delineate a network within the cells. 6. The coupling ratio (CR) between cell pairs measured in dual patch-clamp recordings was 0.356 +/- 0.024. The coupling reversibly decreased to 51 % of the control within 2 min of applying 100 microM ATP. Flash photolysis of 32 microM intracellular caged InsP3 and 1 mM caged Ca2+ reduced CR to 42 and 62 % of the control, respectively. 7. We propose that endolymphatic ATP via P2X and P2Y receptors can control intercellular communication amongst Hensen's cells by reducing gap junction conductance in a Ca2+- and InsP3-dependent manner.

Adenosine Triphosphate↗

PubMed2001

Can you still see the cochlea for the molecules?

It is now established that the mammalian cochlea uses active amplification of incoming sound to achieve sensitivity. Cellular details are emerging slowly. Recent studies of sensory hair cells have highlighted the possible molecular bases for amplification and the components of sensitivity regulation within the cochlea: a synthesis is likely to depend on effective and physiologically informed modelling.

Animals↗

PubMed2000

Molecular mechanisms of sound amplification in the mammalian cochlea.

Mammalian hearing depends on the enhanced mechanical properties of the basilar membrane within the cochlear duct. The enhancement arises through the action of outer hair cells that act like force generators within the organ of Corti. Simple considerations show that underlying mechanism of somatic motility depends on local area changes within the lateral membrane of the cell. The molecular basis for this phenomenon is a dense array of particles that are inserted into the basolateral membrane and that are capable of sensing membrane potential field. We show here that outer hair cells selectively take up fructose, at rates high enough to suggest that a sugar transporter may be part of the motor complex. The relation of these findings to a recent candidate for the molecular motor is also discussed.

Animals↗

PubMed2000

Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the immortomouse.

1. We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release.

Acetylcholine↗

PubMed1999

The fast activating potassium current, I(K,f), in guinea-pig inner hair cells is regulated by protein kinase A.

The mammalian inner hair cell (IHC) responds to displacements produced in the cochlea by sound by releasing neurotransmitter from its basal pole. A basolateral fast activating potassium current, called I(K,f), allows IHCs to act as sensory cells at high frequencies by shortening the membrane time constant. This current is co-expressed with a slower activating current, I(K,s). We have studied the intracellular regulation of IHC currents using the whole-cell patch clamp technique in conjunction with agents that influence the function of protein kinase A (PKA). Bath applied 8-Bromo-cAMP, an activator of PKA, increased the amplitude of outwardly rectifying currents and shortened the exponential time constant of activation. Following blockade of I(K,s) by intracellular 4-AP, I(K,f) could be reduced in amplitude by H-89, an inhibitor of PKA. Our results suggest that PKA regulates I(K,f) and so shapes the frequency response of IHCs.

4-Aminopyridine↗

PubMed1999

Regulation of ionic currents by protein kinase A and intracellular calcium in outer hair cells isolated from the guinea-pig cochlea.

Two prominent potassium currents, termed IK and IK,n, and a cation current are found in outer hair cells (OHCs) of the guinea-pig cochlea. We report here whole-cell recordings which indicate that the currents are regulated by intracellular factors. 8-bromo-cAMP (500 microM), a membrane-permeable cAMP analogue, activated potassium currents in OHCs in both apical and basal turns of the cochlea. In OHCs from the cochlear apex, the drug effect was largest at potentials positive to -40 mV, indicating IK as the target. In short cells from the cochlear base, both IK and IK,n were affected. The effects of 8-bromo-cAMP could be blocked by the presence of 1 microM H-89 (a protein kinase A inhibitor) in the patch pipette solution. Extracellular application of 10 nM okadaic acid, a protein phosphatase inhibitor, also activated both potassium currents. Currents were also modulated by intracellular calcium. IK was activated in long cells by photorelease of calcium from the caged compound nitr5. Cation current activation required calcium release by photolysis of DM-nitrophen, a compound releasing more calcium. The results show that OHC potassium channels are regulated by background phosphorylation through protein kinase A and dephosphorylation by protein phosphatase. Cellular calcium also activates IK and the cation channel, but with different sensitivities.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

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 degrees C and to differentiate at 39 degrees 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 (PK/PNa=2.2) and activated by 10 microM forskolin. These properties identify the slowly activating current as Ih. A proportion of proliferating and differentiating cells exhibited a voltage-gated Na+ current, INa. INa was abolished in Na-free external medium and was inhibited reversibly by tetrodotoxin (TTX) with Ki=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.

Animals↗

PubMed1999

A sugar transporter as a candidate for the outer hair cell motor.

Forces developed by cochlear outer hair cells (OHCs) are responsible for the sharp tuning that underlies sensitivity and frequency selectivity in the ear. OHCs exhibit a voltage-dependent motility involving a 'motor' protein embedded in the basolateral membrane. The motor has so far resisted molecular identification. Here we provide evidence that it may be related to a fructose transporter. We show that OHCs are able to transport this sugar selectively and that the sugar alters electrical properties of the OHC motor. These data can be combined into an integrated model of a sugar carrier, that makes the novel prediction, demonstrated here, that such 'neutral' transporters can be voltage dependent.

Animals↗

PubMed1998

A potassium current in guinea-pig outer hair cells activated by ion channel blocker DCDPC.

Fenamate compounds have been reported to inhibit ion channels in a number of tissues, including a non-selective cation channel in the mammalian outer hair cell (OHC). We have further investigated the effects of 3'-5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) on OHC currents using the whole-cell configuration of the patch clamp technique. Extracellular application of 10 microM DCDPC rapidly and reversibly activated an inward current at hyperpolarized potentials. The DCDPC-activated current appeared in the shorter OHCs from the basal turns of the cochlea. The reversal potential of the inward current was dependent on the external K+ ion concentration. An outwardly rectifying K+ current, found predominantly in OHCs from apical turns, was reversibly inhibited by DCDPC. These results suggest that DCDPC has a significant effect on OHC physiology at all tonotopic locations along the basilar membrane and so may have implications for cochlear function during fenamate intake.

Animals↗

PubMed1998

Auditory hair cell precursors immortalized from the mammalian inner ear.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to differentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least five characteristic hair cell markers. These markers are the transcription factor Brn3.1, the alpha 9 subunit of the acetylcholine receptor, the stereociliary protein fimbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.

Animals↗

PubMed1997

An intrinsic frequency limit to the cochlear amplifier.

Hearing in mammals depends on a feedback process within the inner ear termed the 'cochlear amplifier'. The essential components of this amplifier are sensorimotor cells, the outer hair cells, which transduce motion of the basilar membrane induced by sound and generate forces to cancel the viscous damping of the cochlear partition. Outer hair cells alter the passive mechanics of the cochlea, enhancing both the sensitivity and the frequency selectivity of the auditory system. The molecular basis of the mechanism is thought to be a voltage-sensitive 'motor' protein, as yet unidentified, embedded in the basolateral membrane of the outer hair cell. The cochlear amplifier operates up to at least 22 kHz, but by measuring both the charge and mechanical movements associated with the motor in isolated membrane patches under voltage clamp, we show here that the limiting frequency at which the motor operates lies near 25 kHz. This value therefore sets an upper limit to the range of hearing in mammalian cochleas using this mechanism. The fast charge movement, arising from charge displacement within the presumed motor molecule, further suggests that the protein is more likely to be related to a transporter than to a modified ion channel.

Animals↗

PubMed1997

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 Momega pipettes with tip diameters of 3 micron 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/micron2. 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.

Animals↗

PubMed1997

Evidence for an Na(+)-K(+)-Cl- cotransporter in mammalian type I vestibular hair cells.

In amniotes, there are two types of hair cells, designated I and II, that differ in their morphology, innervation pattern, and ionic membrane properties. Type I cells are unique among hair cells in that their basolateral surfaces are almost completely enclosed by an afferent calyceal nerve terminal. Recently, several lines of evidence have ascribed a motile function to type I hair cells. To investigate this, elevated external K+, which had been used previously to induce hair cell shortening, was used to induce shape changes in dissociated mammalian type I vestibular hair cells. Morphologically identified type I cells shortened and widened when the external K+ concentration was raised isotonically from 2 to 125 mM. The shortening did not require external Ca2+ but was abolished when external Cl- was replaced with gluconate or sulfate and when external Na+ was replaced with N-methyl-D-glucamine. Bumetanide (10-100 microM), a specific blocker of the Na(+)-K(+)-Cl- cotransporter, significantly reduced K(+)-induced shortening. Hyposmotic solution resulted in type I cell shape changes similar to those seen with high K+, i.e., shortening and widening. Type I cells became more spherical in hyposmotic solution, presumably as a result of a volume increase due to water influx. In hypertonic solution, cells became narrower and increased in length. These results suggest that shape changes in type I hair cells induced by high K+ are due, at least in part, to ion and solute entry via an Na(+)-K(+)-Cl- cotransporter, which results in cell swelling. A scheme is proposed whereby the type I hair cell depolarizes and K+ leaves the cell via voltage-dependent K+ channels and accumulates in the synaptic space between the type I hair cell and calyx. Excess K+ could then be removed from the intercellular space by uptake via the cotransporter.

Animals↗

PubMed1996

Differential expression of outer hair cell potassium currents in the isolated cochlea of the guinea-pig.

1. Whole-cell currents were recorded from outer hair cells (OHCs) in undissociated tissues from the organ of Corti. The experiments allowed ionic currents to be measured in cells with precise localization on the three most apical cochlear turns. 2. Two major potassium currents were expressed in the cells. One current, named IK, was half-activated at -24 mV and was most prominent in the most apical turn, turn 4. A second, named IK.n, was half-activated at -92 mV and was the major contributor to the current-voltage (I-V) curve of cells from the more basal turns, turns 3 and 2, of the cochlea. 3. IK was specifically blocked by 100 microM 4-aminopyridine (4-AP). In contrast, IK.n was reduced by 5 mM external barium. Superfusion with zero calcium produced no effect on currents in the range from -60 to 0 mV, but reduced currents by a maximum of 15% outside this range. 4. The cell input conductance increased systematically from 3.4 nS in turn 4 to 40 nS in turn 2 measured at a holding potential of -70 mV. 5. The mean leak conductance, measured from the slope of the I-V curve at -110 mV, decreased systematically from 5.2 nS in turn 2, to 2.9 nS in turn 3 and 2.2 nS in turn 4. 6. These data show that hair cell properties can be determined in undissociated cells and are likely to provide a good estimate of the properties of the cells in the intact cochlea. Differences with the properties of isolated OHCs are discussed.

4-Aminopyridine↗

PubMed1996

Finite element micromechanical modeling of the cochlea in three dimensions.

A new cochlear modeling technique has been developed in which the number of assumptions required in model formulation is significantly less than in previous modeling studies. The main new feature of the method is that it allows individual cellular and membrane components of the organ of Corti to be embedded within the model fluid in their true structural positions, with connections to neighboring elements reflecting anatomical geometry. The cochlea is divided into a three-dimensional finite element (3-D FE) network of nodes, connected by branches representing the local mechanical properties. The model system of simultaneous equations, obtained by applying continuity at each node, is solved iteratively using a variant of the conjugate gradient method. Here the formulation and implementation of the 3-D FE method are described. Force generation by outer hair cells is included and results are presented which demonstrate the effect of tectorial membrane and Deiters' cell mechanical properties on the effectiveness of the cochlear amplifier.

Animals↗