Helen J. Kennedy

Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria.

Increases in the concentration of free ATP within the islet β-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K+(KATP) channels and activating Ca2+ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] in subdomains of single living MIN6 and primary β-cells. Resting [ATP] in the cytosol ([ATP]c), in the mitochondrial matrix ([ATP]m), and beneath the plasma membrane ([ATP]pm) were similar (∼1 mm). Elevations in extracellular glucose concentration (3–30 mm) increased free [ATP] in each domain with distinct kinetics. Thus, sustained increases in [ATP]m and [ATP]pm were observed, but only a transient increase in [ATP]c. However, detectable increases in [ATP]c and [ATP]pm, but not [ATP]m, required extracellular Ca2+. Enhancement of glucose-induced Ca2+ influx with high [K+] had little effect on the apparent [ATP]c and [ATP]m increases but augmented the [ATP]pm increase. Underlying these changes, glucose increased the mitochondrial proton motive force, an effect mimicked by high [K+]. These data support a model in which glucose increases [ATP]m both through enhanced substrate supply and by progressive Ca2+-dependent activation of mitochondrial enzymes. This may then lead to a privileged elevation of [ATP]pm, which may be essential for the sustained closure of KATP channels. Luciferase imaging would appear to be a useful new tool for dynamic in vivo imaging of free ATP concentration.

Force generation by mammalian hair bundles supports a role in cochlear amplification

It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.

Fast adaptation of mechanoelectrical transducer channels in mammalian cochlear hair cells.

Outer hair cells are centrally involved in the amplification and frequency tuning of the mammalian cochlea, but evidence about their transducing properties in animals with fully developed hearing is lacking. Here we describe measurements of mechanoelectrical transducer currents in outer hair cells of rats between postnatal days 5 and 18, before and after the onset of hearing. Deflection of hair bundles using a new rapid piezoelectric stimulator evoked transducer currents with ultra-fast activation and adaptation kinetics. Fast adaptation resembled the same process in turtle hair cells, where it is regulated by changes in stereociliary calcium. It is argued that sub-millisecond transducer adaptation can operate in outer hair cells under the ionic, driving force and temperature conditions that prevail in the intact mammalian cochlea.

The Transduction Channel Filter in Auditory Hair Cells

In the first step in auditory transduction, sound-induced vibrations of the stereociliary bundles on the sensory hair cells are converted into electrical signals by opening of mechanotransducer channels. Faithful transduction and hence auditory performance will be limited by the kinetic properties of these channels. We have measured the time course of mechanotransducer currents in turtle and rat auditory hair cells during rapid deflections of the hair bundle. Current activation in the turtle had a time constant that decreased 10-fold with stimulus amplitude to a limiting value of ∼50 μs. Lowering the external Ca2+ concentration slowed both activation and adaptation time constants. Similar effects were seen in hair cells tuned to low and high frequencies, but the overall kinetics was slower in low-frequency cells. In rat outer hair cells, the time courses of both activation and adaptation were at least 10-fold faster. Although activation kinetics was too fast to characterize accurately, the adaptation time constants in the rat, like the turtle, were Ca2+ dependent and faster in hair cells tuned to higher frequencies. The results imply that mechanotransducer channels operate similarly in turtle and rat but are faster in the mammal to accommodate its higher frequency range of hearing. We suggest that the kinetics of channel activation and adaptation imposes a bandpass filter on transduction, with a center frequency matched to the frequencies detected by the hair cell, which may improve the signal-to-noise ratio near threshold.

Glucose-dependent translocation of insulin promoter factor-1 (IPF-1) between the nuclear periphery and the nucleoplasm of single MIN6 beta-cells.

Using laser-scanning confocal microscopy, we have monitored glucose-induced changes in the subcellular localization of insulin promoter factor-1 (IPF-1) labeled with a c-mycepitope tag. This construct trans-activated the insulin promoter in single living MIN6-β-cells as assessed by luciferase-based promoter analysis. IPF-1.c-myc expression also enhanced the response of the insulin promoter to elevations in extracellular glucose concentration. In the majority (148/235, 63%) of cells maintained at low (3 mm) extracellular glucose concentration, IPF-1.c-myc immunoreactivity was confined to the nuclear periphery. Incubation of cells at stimulatory (30 mm) glucose concentrations caused a rapid redistribution of the chimera to the nucleoplasm (775/958, 81% of cells). By contrast, the irrelevant transcription factor c-Fos, tagged with either c-myc or as a chimera with luciferase, was localized exclusively to the nucleoplasm irrespective of the glucose concentration. Furthermore, IPF-1 extended with the bulky (27 kDa) enhanced green fluorescent protein (EGFP) group was confined largely to the nucleoplasm at all glucose concentrations tested and did not support trans-activation of the insulin promoter by glucose. Movement of endogenous IPF-1 from the nuclear periphery to the nucleoplasm may therefore increase the trans-activational capacity of this factor in native β-cells exposed to high extracellular glucose concentrations.

Depolarization of cochlear outer hair cells evokes active hair bundle motion by two mechanisms.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+‐induced Ca2+ release

Inner hair cells of the mammalian cochlea translate acoustic stimuli into ‘phase‐locked’ nerve impulses with frequencies of up to at least 1 kHz. Little is known about the intracellular Ca2+ signal that links transduction to the release of neurotransmitter at the afferent synapse. Here, we use confocal microscopy to provide evidence that Ca2+‐induced Ca2+ release (CICR) may contribute to the mechanism. Line scan images (2 ms repetition rate) of neonatal mouse inner hair cells filled with the fluorescent indicator FLUO‐3, revealed a transient increase in intracellular Ca2+ concentration ([Ca2+]i) during brief (5–50 ms) depolarizing commands under voltage clamp. The amplitude of the [Ca2+]i transient depended upon the Ca2+ concentration in the bathing medium in the range 0–1.3 mm. [Ca2+]i transients were confined to a region near the plasma membrane at the base of the cell in the vicinity of the afferent synapses. The change in [Ca2+]i appeared uniform throughout the entire basal sub‐membrane space and we were unable to observe hotspots of activity. Both the amplitude and the rate of rise of the [Ca2+]i transient was reduced by external ryanodine (20 μm), an agent that blocks Ca2+ release from the endoplasmic reticulum. Intracellular Cs+, commonly used to record at presynaptic sites, produced a similar effect. We conclude that both ryanodine and intracellular Cs+ block CICR in inner hair cells. We discuss the contribution of CICR to the measured [Ca2+]i transient, the implications for synaptic transmission at the afferent synapse and the significance of its sensitivity to intracellular Cs+.

Real-time imaging of gene expression in single living cells

Recent advances in reporter gene technologies are now allowing us to image gene transcription at the single cell level, using either fluorescence or luminescence microscopy. Here, the basis of these techniques is outlined and their advantages and disadvantages in various biological systems are discussed.

The Resting Transducer Current Drives Spontaneous Activity in Prehearing Mammalian Cochlear Inner Hair Cells

Spontaneous Ca2+-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately −75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca2+ concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately −55 mV). We propose that, in vivo, spontaneous Ca2+ action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.

Regulation of alternative VEGF-A mRNA splicing is a therapeutic target for analgesia.

Vascular endothelial growth factor-A (VEGF-A) is best known as a key regulator of the formation of new blood vessels. Neutralization of VEGF-A with anti-VEGF therapy e.g. bevacizumab, can be painful, and this is hypothesized to result from a loss of VEGF-A-mediated neuroprotection. The multiple vegf-a gene products consist of two alternatively spliced families, typified by VEGF-A165a and VEGF-A165b (both contain 165 amino acids), both of which are neuroprotective. Under pathological conditions, such as in inflammation and cancer, the pro-angiogenic VEGF-A165a is upregulated and predominates over the VEGF-A165b isoform. We show here that in rats and mice VEGF-A165a and VEGF-A165b have opposing effects on pain, and that blocking the proximal splicing event – leading to the preferential expression of VEGF-A165b over VEGF165a – prevents pain in vivo. VEGF-A165a sensitizes peripheral nociceptive neurons through actions on VEGFR2 and a TRPV1-dependent mechanism, thus enhancing nociceptive signaling. VEGF-A165b blocks the effect of VEGF-A165a. After nerve injury, the endogenous balance of VEGF-A isoforms switches to greater expression of VEGF-Axxxa compared to VEGF-Axxxb, through an SRPK1-dependent pre-mRNA splicing mechanism. Pharmacological inhibition of SRPK1 after traumatic nerve injury selectively reduced VEGF-Axxxa expression and reversed associated neuropathic pain. Exogenous VEGF-A165b also ameliorated neuropathic pain. We conclude that the relative levels of alternatively spliced VEGF-A isoforms are critical for pain modulation under both normal conditions and in sensory neuropathy. Altering VEGF-Axxxa/VEGF-Axxxb balance by targeting alternative RNA splicing may be a new analgesic strategy.