Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion ...channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes,
and
, and a
ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology.
PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals
and have essential roles in diverse physiological settings
. In vitro studies have proposed that PIEZO channels ...transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore
. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.
Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn²⁺-sensitive proton ...conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K⁺ current. We identify KIR2.1 as the acid-sensitive K⁺ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of KIR2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K⁺ current in amplifying the sensory response, entry of protons through the Zn²⁺-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K⁺ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.
The conversion of auditory and vestibular stimuli into electrical signals is initiated by force transmitted to a mechanotransduction channel through the tip link, a double stranded protein filament ...held together by two adhesion bonds in the middle. Although thought to form a relatively static structure, the dynamics of the tip-link connection has not been measured. Here, we biophysically characterize the strength of the tip-link connection at single-molecule resolution. We show that a single tip-link bond is more mechanically stable relative to classic cadherins, and our data indicate that the double stranded tip-link connection is stabilized by single strand rebinding facilitated by strong cis-dimerization domains. The measured lifetime of seconds suggests the tip-link is far more dynamic than previously thought. We also show how Ca
alters tip-link lifetime through elastic modulation and reveal the mechanical phenotype of a hereditary deafness mutation. Together, these data show how the tip link is likely to function during mechanical stimuli.
Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the ...receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3-5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.
Significance
Among the five basic tastes, sour is one of the least understood. Notably, the sour receptor remains to be identified, and molecular mechanisms by which sour stimuli are detected are ...largely not known. Previous work has shown that H
+
ions can directly enter sour taste cells, eliciting a change in membrane potential and acidification of the cell cytosol. In the present work, we identify a second element of sensory transduction, a K
+
channel, K
IR
2.1, which is inhibited by intracellular acidification. The presence of an acid-sensitive K
+
channel in sour taste cells allows for amplification of the sensory response and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.
Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn
2+
-sensitive proton conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K
+
current. We identify K
IR
2.1 as the acid-sensitive K
+
channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the
Kcnj2
gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of K
ir
2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K
+
current in amplifying the sensory response, entry of protons through the Zn
2+
-sensitive conductance produces a transient block of the K
IR
2.1 current. The identification in sour taste cells of an acid-sensitive K
+
channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.
Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn2+-sensitive proton ...conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K+ current. We identify KIR2.1 as the acid-sensitive K+ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of KIR2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K+ current in amplifying the sensory response, entry of protons through the Zn2+-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K+ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.
Sour taste is detected by a subset of taste cells on the tongue and palate epithelium that respond to acids with trains of action potentials. Entry of protons through a Zn2+-sensitive proton ...conductance that is specific to sour taste cells has been shown to be the initial event in sour taste transduction. Whether this conductance acts in concert with other channels sensitive to changes in intracellular pH, however, is not known. Here, we show that intracellular acidification generates excitatory responses in sour taste cells, which can be attributed to block of a resting K+ current. We identify KIR2.1 as the acid-sensitive K+ channel in sour taste cells using pharmacological and RNA expression profiling and confirm its contribution to sour taste with tissue-specific knockout of the Kcnj2 gene. Surprisingly, acid sensitivity is not conferred on sour taste cells by the specific expression of KIR2.1, but by the relatively small magnitude of the current, which makes the cells exquisitely sensitive to changes in intracellular pH. Consistent with a role of the K+ current in amplifying the sensory response, entry of protons through the Zn2+-sensitive conductance produces a transient block of the KIR2.1 current. The identification in sour taste cells of an acid-sensitive K+ channel suggests a mechanism for amplification of sour taste and may explain why weak acids that produce intracellular acidification, such as acetic acid, taste more sour than strong acids.
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