Mouse Panx1 Is Dispensable for Hearing Acquisition and Auditory Function

Abstract

Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1−/− mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1−/−), hemizygous (Panx1+/−) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1−/− mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1−/− cochleae. Hearing sensitivity, outer hair cell-based “cochlear amplifier” and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/− and Panx1−/− mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function.

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea

“…Although pannexin 1 channels 71 are thought to mediate ATP release in other cellular systems, [76][77][78][79][80][81] the function of cochlear pannexins remains obscure. 34,[82][83][84] Prior work with the Panx1 −/− strain used here confirmed (i) successful ablation of Panx1 both in the cochlea and in the brain; (ii) normal hearing sensitivity, normal function of the outer hair cell-based "cochlear amplifier" and absence of cochlear nerve defects; (iii) normal expression of inner ear connexins and gap junction communication in the organ of Corti. 34 These published results and the data presented here together rule out that pannexin 1 channels contribute to the purinergic signaling that underlies Ca 2+ wave propagation in the developing cochlea.…”

“…34,[82][83][84] Prior work with the Panx1 −/− strain used here confirmed (i) successful ablation of Panx1 both in the cochlea and in the brain; (ii) normal hearing sensitivity, normal function of the outer hair cell-based "cochlear amplifier" and absence of cochlear nerve defects; (iii) normal expression of inner ear connexins and gap junction communication in the organ of Corti. 34 These published results and the data presented here together rule out that pannexin 1 channels contribute to the purinergic signaling that underlies Ca 2+ wave propagation in the developing cochlea. 2 Gjb6 −/− mice, in which Cx30 is deleted globally 38 and Cx26 is dramatically down-regulated in the sensory epithelium of the developing cochlea, 33 are a model of non-syndromic hearing loss and deafness (DFNB1, reviewed in ref.…”

Double deletion of Panx1 and Panx3 affects skin and bone but not hearing

“…Two-way repeated measures ANOVA with Sidak's post-hoc tests were performed for each individual stimulus. ***p < 0.001, N number of biological replicates the auditory phenotype of the first-generated Panx1 −/− mice and found that they did not have hearing loss [46]. To rule out the possibility that in the global Panx1 KO, Panx3 could be compensating for its function, we tested the dKO hearing and found that both pannexins are dispensable for hearing function.…”

Knockout of Pannexin-1 Induces Hearing Loss

“…Recently, another Panx1 KO mouse line (B6;129- Panx1 tm1.Fam/Cnrm , European Mouse Mutant Archive (EMMA): E11476) [ 32 ] has also been reported to have no hearing loss [ 35 ]. However, it has been reported that the brain tissues of this EMMA Panx1 KO mouse line showed no negative reaction to Panx1 in Western blotting using several anti-Panx1 antibodies [ 36 ], including a chicken anti-human Panx1 antibody used in this study, whose specificity was widely validated in previous experiments by multiple assays in different Panx1 KO mouse lines [ 8 , 9 , 21 , 30 , 37 , 38 ].…”

“…Finally, Panx1 deficiency-induced hearing loss is progressive [ 20 , 30 ]; the increase in ABR threshold is small at postnatal day 30 (P30) and becomes large and apparent after P60 [ 20 , 30 ]. The ABR threshold in the previous report [ 35 ] was averaged from P30 to P90. This could also attenuate the potential difference.…”