Topology of Transmembrane Channel-like Gene 1 Protein

Abstract

Mutations of transmembrane channel-like gene 1 (TMC1) cause hearing loss in humans and mice. TMC1 is the founding member of a family of genes encoding proteins of unknown function that are predicted to contain multiple transmembrane domains. The goal of our study was to define the topology of mouse TMC1 expressed heterologously in tissue culture cells. TMC1 was retained in the endoplasmic reticulum (ER) membrane of five tissue culture cell lines that we tested. We used anti-TMC1 and anti-HA antibodies to probe the topologic orientation of three native epitopes and seven HA epitope tags along full-length TMC1 after selective or complete permeabilization of transfected cells with digitonin or Triton X-100, respectively. TMC1 was present within the ER as an integral membrane protein containing six transmembrane domains and cytosolic N-and Ctermini. There is a large cytoplasmic loop, between the fourth and fifth transmembrane domains, with two highly conserved hydrophobic regions that might associate with or penetrate, but do not span, the plasma membrane. Our study is the first to demonstrate that TMC1 is a transmembrane protein. The topologic organization revealed by this study shares some features with the shaker-TRP superfamily of ion channels. Keywordsdeafness; endoplasmic reticulum; hearing; topology; transmembrane Mutations in transmembrane channel-like gene 1 (TMC1/Tmc1) can cause dominant or recessive hearing loss in humans and mice (1,2). Tmc1 mRNA is specifically expressed in neurosensory hair cells of the inner ear (1,2). Cochlear neurosensory hair cells of Tmc1 mutant mice fail to mature into fully functional sensory receptors (3) and exhibit concomitant structural degeneration that could be a cause or an effect of the maturational defect (2). The molecular and cellular functions of TMC1 protein remain unknown due, at least in part, to in situ expression levels that are prohibitively low for direct biochemical analysis.There are seven additional mammalian TMC paralogs whose structure and function are also unknown. There are no significant sequence similarities of any TMC protein with other proteins of known function. An initial PSORT-II analysis of human and mouse TMC proteins did not detect any N-terminal signal sequences or other trafficking signals, but it did predict that TMC proteins reside in the plasma membrane (4). The TMC proteins are all predicted to contain six to ten transmembrane domains (TMDs) and a novel, conserved * Andrew J. Griffith, MD, PhD, 5 Research Ct. Room 1A13, Rockville, MD 20850. Tel.: 301-496-1960; Fax: 301-402-7580; griffita@nidcd.nih.gov region, which we termed the TMC domain (4). TMHMM2.0 analysis of mouse and human TMC1 predicts cytoplasmically oriented N-and C-termini and six TMDs that are also predicted for the other paralogs (4). Other algorithms such as PSORTII and TopPred predict two to four additional TMDs, for a total of eight to ten TMDs, per TMC homolog (2,5). PROSITE and NetNGlyc identified several TMC sequence sites with varying probabilities of glycos...

Mechanotransduction in mouse inner ear hair cells requires transmembrane channel–like genes

“…However, since structurally visible components of the complex, such as tip links, remain intact (Figure 4), the missing component remains obscure. An alternate hypothesis that would explain our observations is that TMC1 and TMC2 may be components of the mechanotransduction channel itself (1,(3)(4)(5). The topology of TMC1 and TMC2 resembles that of a large superfamily of proteins, including Shaker K + and TRP channels, with 6 membrane-spanning segments and cytoplasmic N and C termini (1,5,35,36).…”

“…An alternate hypothesis that would explain our observations is that TMC1 and TMC2 may be components of the mechanotransduction channel itself (1,(3)(4)(5). The topology of TMC1 and TMC2 resembles that of a large superfamily of proteins, including Shaker K + and TRP channels, with 6 membrane-spanning segments and cytoplasmic N and C termini (1,5,35,36). Moreover, in silico hydropathy analyses imply the existence of two hydrophobic segments that are not predicted to span the membrane and may thus constitute a pore-loop structure (5).…”

“…The function of TMC genes remains obscure, and the in silico translation products have no significant sequence similarity to proteins or domains of known function; however, all are predicted to encode integral membrane proteins with at least 6 membranespanning domains (3,4). This 6-pass transmembrane topology has been experimentally confirmed for mouse TMC1 expressed in heterologous systems (5) and suggests it may function as a receptor, pump, transporter, or channel (3,4).…”

Perception of sound and gravity by TMC1 and TMC2

“…Although the specific functions of the proteins encoded by these genes are unknown, bioinformatic analysis (18) and data obtained from in vitro heterologous systems (19) suggest that they function as membrane channels or transporters (18)(19)(20). Consistent with the genetic data linking TMC1 and TMC2 to a role in hearing, it has been shown that Tmc1 and Tmc2 mRNA is expressed in the inner ear (12,17).…”

Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry

“…Digitonin forms pores in cholesterol-rich membranes such as the plasma membrane, whereas cholesterolpoor membranes such as the endoplasmic reticulum are not affected. Low concentrations of digitonin have been used before to achieve this goal (51)(52)(53). Here, we fixed uninfected HaCaT cells, permeabilized with a high concentration (100 g/ml) and low concentration (5 g/ml) of digitonin.…”