Fabio Mammano

ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear

Extracellular ATP controls various signaling systems including propagation of intercellular Ca2+ signals (ICS). Connexin hemichannels, P2x7 receptors (P2x7Rs), pannexin channels, anion channels, vesicles, and transporters are putative conduits for ATP release, but their involvement in ICS remains controversial. We investigated ICS in cochlear organotypic cultures, in which ATP acts as an IP3-generating agonist and evokes Ca2+ responses that have been linked to noise-induced hearing loss and development of hair cell-afferent synapses. Focal delivery of ATP or photostimulation with caged IP3 elicited Ca2+ responses that spread radially to several orders of unstimulated cells. Furthermore, we recorded robust Ca2+ signals from an ATP biosensor apposed to supporting cells outside the photostimulated area in WT cultures. ICS propagated normally in cultures lacking either P2x7R or pannexin-1 (Px1), as well as in WT cultures exposed to blockers of anion channels. By contrast, Ca2+ responses failed to propagate in cultures with defective expression of connexin 26 (Cx26) or Cx30. A companion paper demonstrates that, if expression of either Cx26 or Cx30 is blocked, expression of the other is markedly down-regulated in the outer sulcus. Lanthanum, a connexin hemichannel blocker that does not affect gap junction (GJ) channels when applied extracellularly, limited the propagation of Ca2+ responses to cells adjacent to the photostimulated area. Our results demonstrate that these connexins play a dual crucial role in inner ear Ca2+ signaling: as hemichannels, they promote ATP release, sustaining long-range ICS propagation; as GJ channels, they allow diffusion of Ca2+-mobilizing second messengers across coupled cells.

Impaired permeability to Ins(1,4,5)P3 in a mutant connexin underlies recessive hereditary deafness.

Connexins are membrane proteins that assemble into gap-junction channels and are responsible for direct, electrical and metabolic coupling between connected cells. Here we describe an investigation of the properties of a recombinantly expressed recessive mutant of connexin 26 (Cx26), the V84L mutant, associated with deafness. Unlike other Cx26 mutations, V84L affects neither intracellular sorting nor electrical coupling, but specifically reduces permeability to the Ca2+-mobilizing messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Both the permeability to Lucifer Yellow and the unitary channel conductance of V84L-mutant channels are indistinguishable from those of the wild-type Cx26. Injection of Ins(1,4,5)P3 into supporting cells of the rat organ of Corti, which abundantly express Cx26, ensues in a regenerative wave of Ca2+ throughout the tissue. Blocking the gap junction communication abolishes wave propagation. We propose that the V84L mutation reduces metabolic coupling mediated by Ins(1,4,5)P3 to an extent sufficient to impair the propagation of Ca2+ waves and the formation of a functional syncytium. Our data provide the first demonstration of a specific defect of metabolic coupling and offer a mechanistic explanation for the pathogenesis of an inherited human disease.

How well do we understand the cochlea

As sensory cells, hair cells within the mammalian inner ear convert sounds into receptor potentials when their projecting stereocilia are deflected. The organ of Corti of the cochlea contains two types of hair cell, inner and outer hair cells, which differ in function. It has been appreciated for over two decades that although inner hair cells act as the primary receptor cell for the auditory system, the outer hair cells can also act as motor cells. Outer hair cells respond to variation in potential, and change length at rates unequalled by other motile cells. The forces generated by outer hair cells are capable of altering the delicate mechanics of the cochlear partition, increasing hearing sensitivity and frequency selectivity. The discovery of such hair-cell motility has modified the view of the cochlea as a simple frequency analyser into one where it is an active non-linear filter that allows only the prominent features of acoustic signals to be transmitted to the acoustic nerve by the inner hair cells. In this view, such frequency selectivity arises through the suppression of adjacent frequencies, a mechanical effect equivalent to lateral inhibition in neural structures. These processes are explained by the interplay between the hydrodynamic interactions among different parts of the cochlear partition and the effective non-linear behaviour of the cell motor.

p53 at the endoplasmic reticulum regulates apoptosis in a Ca2+-dependent manner

Significance Accumulating evidence has underscored the role of cytosolic p53 in promoting cell death. Different reports have revealed that p53 participates in apoptosis induction by acting directly at mitochondria. However, because p53 can mediate apoptosis without its DNA-binding domain (the domain proposed to be fundamental for the targeting of p53 to mitochondria), the mitochondrial localization of p53 is likely not the only transcription-independent mechanism by which p53 promotes apoptosis. Here we demonstrate that p53 at the endoplasmic reticulum (ER) and at mitochondria-associated membranes, interacting with sarco/ER Ca2+-ATPase pumps, modulates ER–mitochondria cross-talk and, in turn, Ca2+-dependent apoptosis. The tumor suppressor p53 is a key protein in preventing cell transformation and tumor progression. Activated by a variety of stimuli, p53 regulates cell-cycle arrest and apoptosis. Along with its well-documented transcriptional control over cell-death programs within the nucleus, p53 exerts crucial although still poorly understood functions in the cytoplasm, directly modulating the apoptotic response at the mitochondrial level. Calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria represents a critical signal in the induction of apoptosis. However, the mechanism controlling this flux in response to stress stimuli remains largely unknown. Here we show that, in the cytoplasm, WT p53 localizes at the ER and at specialized contact domains between the ER and mitochondria (mitochondria-associated membranes). We demonstrate that, upon stress stimuli, WT p53 accumulates at these sites and modulates Ca2+ homeostasis. Mechanistically, upon activation, WT p53 directly binds to the sarco/ER Ca2+-ATPase (SERCA) pump at the ER, changing its oxidative state and thus leading to an increased Ca2+ load, followed by an enhanced transfer to mitochondria. The consequent mitochondrial Ca2+ overload causes in turn alterations in the morphology of this organelle and induction of apoptosis. Pharmacological inactivation of WT p53 or naturally occurring p53 missense mutants inhibits SERCA pump activity at the ER, leading to a reduction of the Ca2+ signaling from the ER to mitochondria. These findings define a critical nonnuclear function of p53 in regulating Ca2+ signal-dependent apoptosis.

A Mechanism for Sensing Noise Damage in the Inner Ear

Our sense of hearing requires functional sensory hair cells. Throughout life those hair cells are subjected to various traumas, the most common being loud sound. The primary effect of acoustic trauma is manifested as damage to the delicate mechanosensory apparatus of the hair cell stereocilia. This may eventually lead to hair cell death and irreversible deafness. Little is known about the way in which noxious sound stimuli affect individual cellular components of the auditory sensory epithelium. However, studies in different types of cell cultures have shown that damage and mechanical stimulation can activate changes in intracellular free calcium concentration ([Ca(2+)](i)) and elicit intercellular Ca(2+) waves. Thus an attractive hypothesis is that changes in [Ca(2+)](i), propagating as a wave through support cells in the organ of Corti, may constitute a fundamental mechanism to signal the occurrence of hair cell damage. The mechanism we describe here exhibits nanomolar sensitivity to extracellular ATP, involves regenerative propagation of intercellular calcium waves due to ATP originating from hair cells, and depends on functional IP(3)-sensitive intracellular stores in support cells.

Biophysics of the cochlea II: Stationary nonlinear phenomenology

Nonlinearities affecting cochlear mechanics produce appreciable compression in the basilar membrane (BM) input/output (I/O) functions at the characteristic frequency for sound-pressure levels (SPLs) as low as 20 dB (re: 20 microPa). This is thought to depend upon saturation of the outer hair cell (OHC) mechanoelectrical transducer (MET). This hypothesis was tested by solving a nonlinear integrodifferential equation that describes the BM vibration in an active cochlea. The equation extends a previously developed linear approach [Mammano and Nobili, J. Acoust. Soc. Am. 93, 3320-3332 (1993)], here modified to include saturating MET, with a few corrections mainly concerning tectorial membrane resonance and OHC coupling to the BM. Stationary solutions were computed by iteration in the frequency domain for a wide range of input SPLs, generating BM I/O functions, frequency response envelopes, and two-tone distortion products. Traveling-wave amplitude envelopes were also computed for a fixed suppressor and several suppressed tones in order to evidence the phenomenon of two-tone suppression (frequency masking) at the mechanical level. All results accord nicely with experimental data.

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.

A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Ca2+ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca2+ pulse. The G293S and G283S mutations delayed the dissipation of Ca2+ transients induced in CHO cells by InsP3. In organotypic cultures, Ca2+ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca2+ transients induced by Ca2+ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.

Coordinated control of connexin 26 and connexin 30 at the regulatory and functional level in the inner ear

Connexin 26 (Cx26) and connexin 30 (Cx30) are encoded by two genes (GJB2 and GJB6, respectively) that are found within 50 kb in the same complex deafness locus, DFNB1. Immunocytochemistry and quantitative PCR analysis of Cx30 KO mouse cultures revealed that Cx26 is downregulated at the protein level and at the mRNA level in nonsensory cells located between outer hair cells and the stria vascularis. To explore connexin coregulation, we manipulated gene expression using the bovine adeno-associated virus. Overexpression of Cx30 in the Cx30 KO mouse by transduction with bovine adeno-associated virus restored Cx26 expression, permitted the formation of functional gap junction channels, and rescued propagating Ca2+ signals. Ablation of Cx26 by transduction of Cx26loxP/loxP cultures with a Cre recombinase vector caused concurrent downregulation of Cx30 and impaired intercellular communication. The coordinated regulation of Cx26 and Cx30 expression appears to occur as a result of signaling through PLC and the NF-κB pathway, because activation of IP3-mediated Ca2+ responses by stimulation of P2Y receptors for 20 min with 20 nM ATP increased the levels of Cx26 transcripts in Cx30 KO cultures. This effect was inhibited by expressing a stable form of the IκB repressor protein that prevents activation/translocation of NF-κB. Thus, our data reveal a Ca2+-dependent control in the expression of inner ear connexins implicated in hereditary deafness as well as insight into the hitherto unexplained observation that some deafness-associated DFNB1 alleles are characterized by hereditable reduction of both GJB2 and GJB6 expression.

Permeability and gating properties of human connexins 26 and 30 expressed in HeLa cells

Human connexins 26 and 30 were expressed either through the bicistronic pIRES-EGFP expression vector or as EYFP-tagged chimeras. When transiently transfected in communication-incompetent HeLa cells, hCx26-pIRES transfectants were permeable to dyes up to 622 Da, but were significantly less permeable to 759 Da molecules. Under the same conditions, permeability of hCx26-EYFP fusion products was comparable to that of hCx26-pIRES, but with significant increase in diffusion at 759 Da, possibly as a consequence of having selected large fluorescent junctional plaques. Dye transfer was limited to 457 Da in hCx30-EYFP transfectants. When reconstructed from confocal serial sections, fluorescent plaques formed by hCx26-EYFP and hCx30-EYFP appeared irregular, often with long protrusions or deep invagination. Similar plaques were observed following immunostaining both in cells transfected with hCx26-pIRES and in HeLa cells stably transfected with mouse Cx26. Tissue conductance (Tg(j)) displayed significantly smaller values (28.8+/-1.8 nS) for stably transfected mCx26 than transiently transfected hCx26 (43.5+/-3.3 nS). These differences reflected in distinct functional dependence of normalized junctional conductance (G(j)) on transjunctional voltage (V(j)). The half-activation voltage for G(j) was close to +/-95 and +/-58 mV in mCx26 and hCx26, respectively. The corresponding parameters for hCx30 transfectants were Tg(j)= 45.2 +/- 3.5 nS and V(0)= +/- 34 mV. These results highlight unexpected differences between mCx26 and hCx26 in this expression system, reinforce the concept that channel permeability may be related to Cx level expression, and indicate that fusion of hCx30 to GFP colour mutants produces channels that are suitable for permeability and gating studies.