Roles of the A and C sites in the manganese-specific activation of MntR.

The manganese transport regulator (MntR) represses the expression of genes involved in manganese uptake in Bacillus subtilis. It selectively responds to Mn(2+) and Cd(2+) over other divalent metal cations, including Fe(2+), Co(2+), and Zn(2+). Previous work has shown that MntR forms binuclear complexes with Mn(2+) or Cd(2+) at two binding sites, labeled A and C, that are separated by 4.4 Å. Zinc activates MntR poorly and binds only to the A site, forming a mononuclear complex. The difference in metal binding stoichiometry suggested a mechanism for selectivity in MntR. Larger metal cations are strongly activating because they can form the binuclear complex, while smaller metal ions cannot bind with the geometry needed to fully occupy both metal binding sites. To investigate this hypothesis, structures of MntR in complex with two other noncognate metal ions, Fe(2+) and Co(2+), have been determined. Each metal forms a mononuclear complex with MntR with the metal ion bound in the A site, supporting the conclusions drawn from the Zn(2+) complex. Additionally, we investigated two site-specific mutants of MntR, E11K and H77A, that contain substitutions of metal binding residues in the A site. While metal binding in each mutant is significantly altered relative to that of wild-type MntR, both mutants retain activity and selectivity for Mn(2+) in vitro and in vivo. That observation, coupled with previous studies, suggests that the A and C sites both contribute to the selectivity of MntR.

Morphological correlates of hearing loss after cochlear implantation and electro-acoustic stimulation in a hearing-impaired Guinea pig model.

Hybrid or electro-acoustic stimulation (EAS) cochlear implants (CIs) are designed to provide high-frequency electric hearing together with residual low-frequency acoustic hearing. However, 30-50% of EAS CI recipients lose residual hearing after implantation. The objective of this study was to determine the mechanisms of EAS-induced hearing loss in an animal model with high-frequency hearing loss. Guinea pigs were exposed to 24 h of noise (12-24 kHz at 116 dB) to induce a high-frequency hearing loss. After recovery, two groups of animals were implanted (n = 6 per group), with one group receiving chronic acoustic and electric stimulation for 10 weeks, and the other group receiving no stimulation during this time frame. A third group (n = 6) was not implanted, but received chronic acoustic stimulation. Auditory brainstem responses were recorded biweekly to monitor changes in hearing. The organ of Corti was immunolabeled with phalloidin, anti-CtBP2, and anti-GluR2 to quantify hair cells, ribbons and post-synaptic receptors. The lateral wall was immunolabeled with phalloidin and lectin to quantify stria vascularis capillary diameters. Bimodal or trimodal diameter distributions were observed; the number and location of peaks were objectively determined using the Aikake Information Criterion and Expectation Maximization algorithm. Noise exposure led to immediate hearing loss at 16-32 kHz for all groups. Cochlear implantation led to additional hearing loss at 4-8 kHz; this hearing loss was negatively and positively correlated with minimum and maximum peaks of the bimodal or trimodal distributions of stria vascularis capillary diameters, respectively. After chronic stimulation, no significant group changes in thresholds were seen; however, elevated thresholds at 1 kHz in implanted, stimulated animals were significantly correlated with decreased presynaptic ribbon and postsynaptic receptor counts. Inner and outer hair cell counts did not differ between groups and were not correlated with threshold shifts at any frequency. As in the previous study in a normal-hearing model, stria vascularis capillary changes were associated with immediate hearing loss after implantation, while little to no hair cell loss was observed even in cochlear regions with threshold shifts as large as 40-50 dB. These findings again support a role of lateral wall blood flow changes, rather than hair cell loss, in hearing loss after surgical trauma, and implicate the endocochlear potential as a factor in implantation-induced hearing loss. Further, the analysis of the hair cell ribbons and post-synaptic receptors suggest that delayed hearing loss may be linked to synapse or peripheral nerve loss due to stimulation excitotoxicity or inflammation. Further research is needed to separate these potential mechanisms of delayed hearing loss.