Postnatal developmental expression of the PDZ scaffolds Na+-H+ exchanger regulatory factors 1 and 2 in the rat cochlea

Abstract

Sensory transduction in the mammalian cochlea requires the maintenance of specialized fluid compartments with distinct ionic compositions. This is achieved by the concerted action of diverse ion channels and transporters, some of which can interact with the PDZ scaffolds, Na + -H + exchanger regulatory factors 1 and 2 (NHERF-1, NHERF-2). Here, we report that NHERF-1 and NHERF-2 are widely expressed in the rat cochlea, and that their expression is developmentally regulated. Reverse transcription/polymerase chain reaction (RT-PCR) and Western blotting initially confirmed the RNA and protein expression of NHERFs. We then performed immunohistochemistry on cochlea during various stages of postnatal development. Prior to the onset of hearing (P8), NHERF-1 immunolabeling was prominently polarized to the apical membrane of cells lining the endolymphatic compartment, including the stereocilia and cuticular plates of the inner and outer hair cells, marginal cells of the stria vascularis, Reissner's epithelia, and tectorial membrane. With maturation (P21, P70), NHERF-1 immunolabeling was reduced in the above structures, whereas labeling increased in the apical membrane of the interdental cells of the spiral limbus and the inner and outer sulcus cells, Hensen's cells, the inner and outer pillar cells, Deiters cells, the inner border cells, spiral ligament fibrocytes, and spiral ganglion neurons (particularly type II). NHERF-1 expression in strial basal and intermediate cells was persistent. NHERF-2 immunolabeling was similar to that for NHERF-1 during postnatal development, with the exception of expression in the synaptic regions beneath the outer hair cells. NHERF-1 and NHERF-2 co-localized with glial fibrillary acidic protein and vimentin in glia. The cochlear localization of NHERF scaffolds suggests that they play important roles in the developmental regulation of ion transport, homeostasis, and auditory neurotransmission.

Na+–H+ exchanger regulatory factor 1 is a PDZ scaffold for the astroglial glutamate transporter GLAST

“…The specificities of the NHERF1 and NHERF2 antibodies for their targets in immunocytochemistry have been demonstrated previously (Kanjhan et al, 2006). We also tested the specificity of the NHERF1 and NHERF2 antibodies on immunoblots of electrophorectically separated SDS-solubilized brain lysate.…”

“…We have recently reported that NHERF1/2 are widely distributed in the cochlea. In particular, NHERF2 immunolabeling was also evident in walls of large blood vessels including spiral modiolar artery and vein (Kanjhan et al, 2006). While the precise function(s) of NHERF2 within endothelial cells remains to be determined, it is interesting to note that several members of the previously characterized NHERF2 binding partners, namely P2Y1 receptor (Fam et al, 2005), PTH receptor (Mahon et al, 2002), and β 2 AR (Hall et al, 1998) are also expressed in endothelial cells of the mammalian brain.…”

“…Antibodies against NHERF1 and NHERF2 (Kanjhan et al, 2006), GLAST (Pow and Barnett, 1999), and GLT1b (Reye et al, 2002) have been described previously. Antibody specific for ezrin (residues 362-585 of human ezrin, GenBank ™ accession number NM_003379) was purchased from Zymed Laboratories (San Francisco, CA, USA).…”

“…NHERF1/2 nucleates the formation of macromolecular complexes by physically linking plasma membrane proteins to the cytoskeleton and recruiting signaling molecules such as PKA (Yun et al, 1997), PKC (Liedtke et al, 2002), and PLC β (Tang et al, 2002) to the complex. Although it was first thought to be restricted to transporting epithelia, NHERF1/2 have since been reported in cochlea (Kanjhan et al, 2006), retina (Nawrot et al, 2004), dorsal root ganglia (Deval et al, 2006), vascular smooth muscle, lymphocytes, and endometrium (Stemmer-Rachamimov et al, 2001). …”

The two-pore domain K+ channel TASK-1 is closely associated with brain barriers and meninges

“…l Extensive TASK-1-IR in glial cells and their processes ensheated at the walls of small to medium-sized blood vessels located deep inside the brain parenchyma in the medullary median septum. Abbreviations: inferior cerebellar peduncle (icp), spinal trigeminal tract (Sp5) c procedures were modified from our previous studies (Kanjhan et al 2004a(Kanjhan et al , 2006. After intracardiac perfusion with heparinized 0.9% NaCl solution containing 1.45 mM NaNO 2 , the brains were fixed in situ with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4).…”