Functional mechanoelectrical transduction (MET) channels of cochlear hair cells require the presence of transmembrane channel-like protein isoforms TMC1 or TMC2. We show that TMCs distinctively influence channel properties. TMC1-dependent channels have larger single-channel conductance, faster adaptation and, in outer hair cells (OHCs), support a tonotopic apex-to-base gradient in channel conductance. The MET channel has a high permeability to calcium which is reduced in two different Tmc1 mutations associated with autosomal dominant deafness. Each MET channel complex exhibits multiple conductance states in ~50 pS increments, basal MET channels having more large-conductance levels. Using mice expressing fluorescently tagged TMCs, we show a three-fold increase in number of TMC1 molecules per stereocilium tip from cochlear apex to base, mirroring the channel conductance gradient in OHCs. The results suggest there are varying numbers of channels per MET complex, each requiring multiple TMC1 molecules, and together operating in a coordinated manner.