1,721,029 research outputs found
Calcium Dynamics in Inner Ear Health and Disease
No full textCa2+ acts as a fundamental signal transduction element in the inner ear, delivering information about sound acceleration and gravity through a small number of mechano-transduction channels in the hair cell stereocilia as far as to the ribbon synapse, where it drives neurotransmission. The genetic approach is proving fundamental in unravelling the molecular basis of Ca2+ function in the control of these key cellular processes. Ablation or missense mutations of the PMCA2 Ca2+-pump of stereocilia cause deafness and loss of balance. To investigate the physiological significance of these genetic defects, we studied PMCA2 Ca2+-extrusion in hair cells of utricle organotypic cultures from neonatal mice inner ear. Confocal Ca2+ imaging showed that the dissipation of stereociliary Ca2+ transients, induced by cytosolic photoliberation, was compromised by various degrees in PMCA2 knockout mice as well as in the mutant deafwaddler and Oblivion mice. Alteration of the intracellular Ca2+ concentration ( ) can trouble the finely tuned control mechanisms of signal transduction, thus resulting as a fundamental physiological parameter to be investigated in the comprehension of deafness mechanisms. By comparing our experimental fluorescence data with those derived from Monte Carlo numerical simulations, we provided a novel method to effectively deconvolve within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. Data analysis performed within this environment indicates that changes of hair cell basolateral during synaptic transmission are primarily controlled by the endogenous Ca2+ buffers at both short (< 1 micron) and long (tens of microns) distances from the presynaptic active zones. Furthermore, we provided quantitative estimates of concentration and kinetics of the endogenous Ca2+-buffers and Ca2+-ATPases in frog vestibular hair cells. We successfully applied mathematical models also in the study of channel permeability to second messengers of gap junctions, intercellular channels connecting supporting cells of the organ of Corti. In particular, it’s known that defective permeation of cAMP or inositol 1,4,5-trisphosphate through gap junction channels is associated with peripheral neuropathies and deafness, respectively. Our model permits quantification of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signaling pathways
What's the Function of Connexin 32 in the Peripheral Nervous System?
Full text linkConnexin 32 (Cx32) is a fundamental protein in the peripheral nervous system (PNS) as its mutations cause the X-linked form of Charcot-Marie-Tooth disease (CMT1X), the second most common form of hereditary motor and sensory neuropathy and a demyelinating disease for which there is no effective therapy. Since mutations of the GJB1 gene encoding Cx32 were first reported in 1993, over 450 different mutations associated with CMT1X including missense, frameshift, deletion and non-sense ones have been identified. Despite the availability of a sizable number of studies focusing on normal and mutated Cx32 channel properties, the crucial role played by Cx32 in the PNS has not yet been elucidated, as well as the molecular pathogenesis of CMT1X. Is Cx32 fundamental during a particular phase of Schwann cell (SC) life? Are Cx32 paired (gap junction, GJ) channels in myelinated SCs important for peripheral nerve homeostasis? The attractive hypothesis that short coupling of adjacent myelin layers by Cx32 GJs is required for efficient diffusion of K+ and signaling molecules is still debated, while a growing body of evidence is supporting other possible functions of Cx32 in the PNS, mainly related to Cx32 unpaired channels (hemichannels), which could be involved in a purinergic-dependent pathway controlling myelination. Here we review the intriguing puzzle of findings about Cx32 function and dysfunction, discussing possible directions for future investigation
Measuring Connexin Hemichannel Opening in Response to an InsP3-Mediated Cytosolic Ca2+ Increase
No full text: The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction
Defects in the ATP2B2 gene causing hereditary hearing and balance loss in mice and humans: a biophysical study of normal and mutated PMCA2 pump function.
No full textCa2+ acts as a fundamental signal transduction element in the inner ear, delivering information about sound acceleration and gravity through a small number of mechano-transduction channels in the hair cell stereocilia as far as to the ribbon synapse, where it drives neurotransmission. The genetic approach is proving fundamental in unravelling the molecular basis of these important biological functions. In particular, ablation or missense mutations of the PMCA2 Ca2+-pump of stereocilia cause deafness and loss of balance. To investigate the physiological significance of these genetic defects, we used a combination of confocal fluorescence microscopy and cytosolic Ca2+ photoliberation. The study of Ca2+-extrusion in hair cells from neonatal mice inner ear permitted us to show that Ca2+ extrusion was compromised by various degrees in PMCA2 knockout mice as well as in the mutant deafwaddler and Oblivion mice. We suggest that the consequent reduced endolymphatic Ca2+ concentration can trouble the finely tuned control mechanisms of signal transduction, eventually resulting in hair cell death
A biophysical approach to the study of the structure and function of connexin channel nanopores.
No full textObjective: Connexins are transmembrane proteins that form intercellular junctional channels in vertebrates and are known or suspected to be involved in a wide variety of biological processes including cardiac development and function, hearing, haematopoesis, regeneration, lens transparency, fertility, immune system function and protection from oxidative stress. Connexin mutations can cause developmental and physiological defects, and link to various diseases. In particular, defective permeation of cAMP or inositol-1,4,5-trisphosphate (InsP 3 ) through connexin channels is associated with peripheral neuropathies and deafness, respectively. Here we present a method to estimate the permeability of single-gap junction channels to second messengers. Study design : Using HeLa cells that overexpressed wild-type human connexin 26 (HCx26wt) as a model system, we combined measurements of junctional conductance and fl uorescence resonance energy transfer (FRET) emission ratio of biosensors selective for cAMP and InsP 3 . Results: The unitary permeabilities to cAMP (47 ± 15 ×10–3 μm3/s) and InsP3 (60 ± 12 ×10–3 μm3/s) were similar, but substantially larger than the unitary permeability to lucifer yellow (LY; 7 ± 3 ×10–3 μm3/s), an exogenous tracer. Conclusion: This method permits quantifi cation of defects of metabolic coupling and can be used to investigate interdependence of intercellular diffusion and cross-talk between diverse signalling pathways
Ca2+ dynamics in auditory and vestibular hair cells: Monte Carlo simulations and experimental results.
No full textWe developed a simulation code in the Matlab environment for the study, using the Monte Carlo method, of cellular phenomena involving diffusion, buffering, extrusion and release of Ca2+. In particular we simulated the entry of Ca2+ at individual presynaptic active zones (hotspots) of auditory and vestibular hair cells, where Ca2+ plays a fundamental role in the transduction of mechanical stimuli, due to sound or acceleration, into electrical signals to be sent to the brain. The realistic reconstruction, in three dimensions, of the cellular boundaries and the derivation of the virtual fluorescence ratio ΔF/F0 (equivalent to the one computed from fluorescence microscopy experiments) allowed us (i) to directly compare simulations to experimental data, (ii) to supply an estimate of the equivalent concentration of Ca2+ reactants (buffers) and (iii) to show how the mass action law hypothesis brakes down because of the local non equilibrium of the system
Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution
No full textSignal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+),sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvotve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (< 1 mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca(2+) dynamics in diverse biological systems. (C) 2007 Elsevier Ltd. All rights reserved
PMCA2 pump mutations and hereditary deafness
No full textHair cells of the inner ear detect sound stimuli, inertial or gravitational forces by deflection of their apical
stereocilia. A small number of stereociliary cation-selective mechanotransduction (MET) channels admit K+ and
Ca2+ ions into the cytoplasm promoting hair cell membrane depolarization and, consequently, neurotransmitter
release at the cell basolateral pole. Ca2+ influx into the stereocilia compartment is counteracted by the unusual
w/a splicing variant of plasma-membrane calcium-pump isoform 2 (PMCA2) which, unlike other PMCA2 variants,
increases only marginally its activity in response to a rapid variation of the cytoplasmic free Ca2+ concentration
([Ca2+]c). Missense mutations of PMCA2w/a cause deafness and loss of balance in humans. Mouse
models in which the pump is genetically ablated or mutated show hearing and balance impairment, which
correlates with defects in homeostatic regulation of stereociliary [Ca2+]c, decreased sensitivity of mechanotransduction
channels to hair bundle displacement and progressive degeneration of the organ of Corti. These
results highlight a critical role played by the PMCA2w/a pump in the control of hair cell function and survival,
and provide mechanistic insight into the etiology of deafness and vestibular disorders
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