Corey, David P.

Gaudet, Rachelle

Sotomayor, Marcos

Elsevier - Publisher Connector 

Molecular Mechanisms of Deafness Mutations Disrupting Tip-Link Function in Hair-Cell Mechanotransduction 

Tuesday, February 18, 2014 449aWT-MscS and the alanine mutants have been undertaken to experimentally test
the predictions from the modelling studies.
References:
1. Martinac B, Curr Top Membr. 2007 58, 25.
2. Nomura T, Cranfield CG, Deplazes E, Owen DM, Macmillian A, Battle AR,
Constantine M, Sokabe M, Martinac B. PNAS 2012, 109, 8770.
3. Cox, C., Nomura, T., Ziegler, CS, Campbell AK, Wann KT, Martinac B. Na-
ture Communications 2013, 4, 2137
2268-Plat
Novel Mutations in the Extracellular Cap of the Mammalian Mechanosen-
sitive Channel TREK-1
Paul Blount, Robin Wray, Irene R. Iscla.
Physiology, U.T. Southwestern Med. Ctr., Dallas, TX, USA.
The Twik related potassium channel 1 (TREK-1) is one of the best studied me-
chanosensitive mammalian channels. TREK-1 is known to play very important
roles in depression, ischemia and vasoregulation. TREK-1 belongs to the fam-
ily of background or "leak" potassium channels are constitutively open at rest
and have a central role in the tuning of neuronal resting membrane potential,
duration of action potential and regulation of neurotransmitter release. These
Kþ channels are members of the family of K2P channels subunits containing
four transmembrane and two pore domains. The functional channel is formed
by two subunits and is predicted to have a two-fold symmetry.
Here we show the feasibility of the use of microbial genetics to study the
structure-function relationship of this mammalian channel. The advantage of
this approach is that we can directly screen for channels with altered pheno-
types and correlate this altered function with structural changes. We have suc-
cessfully expressed a functional TREK-1 channel in bacterial cells, and show
that it can partially rescue the slow growth phenotype of an E .coli strain defi-
cient in three mayor potassium transporters. Furthermore, using random muta-
genesis and bacterial screens we have isolated five mutants that better
remediate the potassium deficiency of this bacterial strain. Because these mu-
tants clustered in a stretch of 20 amino acids in the extracellular cap of the
TREK-1, we think that we have found a functional "hot spot" by utilizing
the power of bacterial genetics.
2269-Plat
Omega 6 Polyunsaturated Fatty Acid-Containing Phospholipids Enhance
Neuronal Cell Mechanics and Touch in C. Elegans
Valeria Vasquez, Michael Krieg, Dean Lockhead, Miriam B. Goodman.
Stanford University, Stanford, CA, USA.
Mechano-electrical transduction (MeT) channels embedded in neuronal cell
membranes are essential for touch and proprioception. Little is understood
about the interplay between nativeMeT channels and membrane phospholipids,
in part because few techniques are available for altering plasma membrane
composition in vivo. Here, we leverage genetic dissection, chemical comple-
mentation, and optogenetics to establish that arachidonic acid (AA), an omega
6 polyunsaturated fatty acid (PUFA), enhances touch sensation and mechanoe-
lectrical transduction activity while incorporated into membrane phospholipids
in C. elegans touch receptor neurons (TRNs). We found that arachidonic acid
acts cell autonomously, since we show that enzymes needed for its synthesis
are expressed in TRNs. We also established that the membrane viscoelastic
properties of TRNs lacking omega 6 PUFAs are altered (i.e., membrane
bending and viscosity), yielding less flexible membranes than wild type, as
determined by atomic force microscopy (AFM) based single-tether extrusion.
Our data suggest that the defect in touch sensation likely reflects a loss of me-
chanotransduction rather than lack of excitability or downstream signaling.
These findings establish that polyunsaturated phospholipids are crucial determi-
nants of both the biochemistry and mechanics of mechanoreceptor neurons and
reinforce the idea that sensory mechanotransduction in animals relies on a
cellular machine composed of both proteins and membrane lipids.
2270-Plat
Touch Activates Mechanosensitive Ion Channels in Merkel Cells In Vitro
Masashi Nakatani1,2, Aislyn M. Nelson3, Ellen A. Lumpkin1,4.
1Department of Dermatology, Columbia University Medical Center, New
York, NY, USA, 2Keio University, Yokohama, Japan, 3Baylor College of
Medicine, Houston, TX, USA, 4Department of Physiology and Cellular
Biophysics, Columbia University, New York, NY, USA.
Merkel cell-neurite complexes are gentle touch receptors that mediate slowly
adapting type I (SAI) responses. Since their description in 1875, Merkel cells
have been proposed to be mechanosensory cells that transduce mechanical
stimuli into electrical signals that activate somatosensory neurons. Consistent
with this model, conditional knockout mice that lack Merkel cells show the
loss of touch-evoked SAI responses. Moreover, in vitro studies on cultured
Merkel cells report calcium elevation in Merkel cells in response to swellingor membrane stretch. Previous studies support the contribution of Merkel cells
to touch sensation, however, the central question of whether Merkel cells are
intrinsically touch sensitive is unanswered. To tackle this problem, we per-
formed live-cell imaging and electrophysiological recordings from mouse Mer-
kel cells. Merkel cells were dissociated from the epidermal skin of transgenic
Atoh1/nGFP mice, whose Merkel cells selectively express green fluorescent
protein. GFP-positive cells were purified using fluorescence-activated cell sort-
ing and cultured for 1-5 days. Individual Merkel cells were stimulated by fam-
ilies of displacements (&0.3-mm steps) with a glass probe driven by a
piezoelectric actuator. Touch-evoked responses were monitored with either ra-
tiometric calcium imaging or tight-seal, whole-cell recordings. Merkel cells
displayed reversible calcium responses to focal displacements applied to
somata. Moreover, electrophysiological recordings demonstrated mechanically
activated inward currents at a negative holding potential. Peak inward currents
ranged from 107.3-431.3 pA. The 10-90% operating range of these mechani-
cally activated currents was 1.750.1 mm (N=10 cells, mean5SE). Like me-
chanosensitive currents in hair cells and somatosensory neurons, Merkel-cell
currents adapted exponentially to sustained stimuli. Quantitative PCR indicated
that Merkel cells expressed both Piezo1 and Piezo2 genes. Together, these data
demonstrate that Merkel cells are intrinsically mechanosensitive in the absence
of other skin cells or somatosensory neurons.
2271-Plat
Molecular Mechanisms of Deafness Mutations Disrupting Tip-Link Func-
tion in Hair-Cell Mechanotransduction
Marcos Sotomayor1, Rachelle Gaudet2, David P. Corey3.
1The Ohio State University, Columbus, OH, USA, 2Harvard University,
Boston, MA, USA, 3Harvard Medical School, Boston, MA, USA.
Hair-cell tip links are fine filaments that directly convey mechanical force to
inner ear mechanotransduction channels. These filaments are made of
protocadherin-15 (PCDH15) and cadherin-23 (CDH23), two deafness-related
proteins that feature long extracellular domains interacting tip-to-tip in a cal-
cium dependent manner. Here we combine X-ray crystallography, molecular
dynamics simulations, and binding experiments to explore the molecular mech-
anisms by which deafness mutations disrupt tip-link function in hair-cell me-
chanotransduction. We find that these mutations disrupt tip links through
impaired interaction between PCDH15 and CDH23 (PCDH15-R113G and
PCDH15-I108N), impaired calcium binding (CDH23-D1010G), subtle weak-
ening of structural stability (CDH23-S47P), or impaired folding (PCDH15-
D157G). Interestingly, the biochemical effects of each of these deafness muta-
tions correlate with the severity of the reported inner-ear phenotype. Our results
shed light on the molecular mechanisms of hair-cell sensory transduction and
may help develop tailored treatments for cadherin-mediated deafness.
Platform: Force Spectroscopy
2272-Plat
Disulfide Bonds are Allosteric Regulator of Mechanical Stability
David Giganti1, Guillaume Stirnemann2, Kevin Yan3, Bruce Berne2,
Julio Fernandez3.
1Columbia University, New York, NY, USA, 2Chemistry, Columbia
University, New York, NY, USA, 3Biological Sciences, Columbia
University, New York, NY, USA.
Disulfide bonds are known to stabilize proteins against perturbations such as
temperature or denaturants. Since mechanical force is the most common protein
denaturant in vivo, there is increasing interest in the role that disulfide bonds
have in the mechanical unfolding of proteins. For instance, disulfide bonds
reduce the contour length of stretched proteins by limiting the extensibility
up to the covalently linked cysteines. However very little is known about the
effect of native disulfide bonds in the strength of structural clamps that deter-
mine mechanical stability.
Here, we use single molecule atomic force spectroscopy to study the mechan-
ical effects of disulfide bonding in immunoglobulin domains (Ig) that are
constitutively under force.
We observe that the formation of native disulfide bonds in immunoglobulin do-
mains triggers drastic differences in the rupture forces, although the two
cognate cysteines do not link together the b-strands of the mechanical clamp
motif. In the case of bacterial gram-negative pilin FimH, disulfide increases
the most probable unfolding force from 297 pN to 425 pN at 400nm/s. In
contrast, the disulfide in the 69th immunoglobulin domain of human titin de-
creases the unfolding force at a pulling rate of 1200nm/s from 271 pN to 195
pN. We observe that both in titin and in the Fim pilus, the disulfide bonds
are remarkably conserved along the entire stretched molecular architecture.
Hence, many Igs in elastic segment of titin and in all Fim Igs are predicted
to change mechanical stability depending of their oxidation state. Our results


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Molecular Mechanisms of Deafness Mutations Disrupting Tip-Link Function in Hair-Cell Mechanotransduction

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