Xenopus TRPN1 (NOMPC) localizes to microtubule-based cilia in epithelial cells, including inner-ear hair cells.

In vertebrates, the senses of hearing and balance depend on hair cells, which transduce sounds with their hair bundles, containing actin-based stereocilia and microtubule-based kinocilia. A longstanding question in auditory science is the identity of the mechanically sensitive transduction channel of hair cells, thought to be localized at the tips of their stereocilia. Experiments in zebrafish implicated the transient receptor potential (TRP) channel NOMPC (drTRPN1) in this role; TRPN1 is absent from the genomes of higher vertebrates, however, and has not been localized in hair cells. Another candidate for the transduction channel, TRPA1, apparently is required for transduction in mammalian and nonmammalian vertebrates. This discrepancy raises the question of the relative contribution of TRPN1 and TRPA1 to transduction in nonmammalian vertebrates. To address this question, we cloned the TRPN1 ortholog from the amphibian Xenopus laevis, generated an antibody against the protein, and determined the protein's cellular and subcellular localization. We found that TRPN1 is prominently located in lateral-line hair cells, auditory hair cells, and ciliated epidermal cells of developing Xenopus embryos. In ciliated epidermal cells TRPN1 staining was enriched at the tips and bases of the cilia. In saccular hair cells, TRPN1 was located prominently in the kinocilial bulb, a component of the mechanosensory hair bundles. Moreover, we observed redistribution of TRPN1 upon treatment of hair cells with calcium chelators, which disrupts the transduction apparatus. This result suggests that although TRPN1 is unlikely to be the transduction channel of stereocilia, it plays an essential role, functionally related to transduction, in the kinocilium.

A family of acid-sensing ion channels from the zebrafish: widespread expression in the central nervous system suggests a conserved role in neuronal communication.

Acid-sensing ion channels (ASICs) are excitatory receptors for extracellular H(+). Proposed functions include synaptic transmission, peripheral perception of pain, and mechanosensation. Despite the physiological importance of these functions, the precise role of ASICs has not yet been established. In order to increase our understanding of the physiological role and basic structure-function relationships of ASICs, we report here the cloning of six new ASICs from the zebrafish (zASICs). zASICs possess the basic functional properties of mammalian ASICs: activation by extracellular H(+), Na(+) selectivity, and block by micromolar concentrations of amiloride. The zasic genes are broadly expressed in the central nervous system, whereas expression in the peripheral nervous system is scarce. This pattern suggests a predominant role for zASICs in neuronal communication. Our results suggest a conserved function for receptors of extracellular H(+) in the central nervous system of vertebrates.

A new subunit of the epithelial Na+ channel identifies regions involved in Na+ self-inhibition.

The epithelial Na+ channel (ENaC) is the apical entry pathway for Na+ in many Na+-reabsorbing epithelia. ENaC is a heterotetrameric protein composed of homologous alpha, beta, and gamma subunits. Mutations in ENaC cause severe hypertension or salt wasting in humans; and consequently, ENaC activity is tightly controlled. According to the concept of Na+ self-inhibition, the extracellular Na+ ion itself can reduce ENaC activity. The molecular basis for Na+ self-inhibition is unknown. Here, we describe cloning of a new ENaC subunit from Xenopus laevis (epsilonxENaC). epsilonxENaC can replace alphaxENaC and formed functional, highly selective, amiloride-sensitive Na+ channels when coexpressed with betaxENaC and gammaxENaC. Channels containing epsilonxENaC showed strong inhibition by extracellular Na+. This Na+ self-inhibition was significantly slower than for alphaxENaC-containing channels. Using site-directed mutagenesis, we show that the proximal part of the large extracellular domain controls the speed of self-inhibition. This suggests that this region is involved in conformational changes during Na+ self-inhibition.