Abstract: | The cytoplasmic amino terminus of HCN1, the primary full-length HCN isoform
expressed in trout saccular hair cells, was found by yeast two-hybrid
protocols to bind the cytoplasmic carboxyl-terminal domain of a protocadherin
15a-like protein. HCN1 was immunolocalized to discrete sites on saccular hair
cell stereocilia, consistent with gradated distribution expected for tip link
sites of protocadherin 15a. HCN1 message was also detected in cDNA libraries
of rat cochlear inner and outer hair cells, and HCN1 protein was
immunolocalized to cochlear hair cell stereocilia. As predicted by the trout
hair cell model, the amino terminus of rat organ of Corti HCN1 was found by
yeast two-hybrid analysis to bind the carboxyl terminus of protocadherin 15
CD3, a tip link protein implicated in mechanosensory transduction. Specific
binding between HCN1 and protocadherin 15 CD3 was confirmed with pull-down
assays and surface plasmon resonance analysis, both predicting dependence on
Ca2+. In the presence of calcium chelators, binding between HCN1
and protocadherin 15 CD3 was characterized by a KD = 2.39
× 10-7 m. Ca2+ at 26.5-68.0
μm promoted binding, with KD = 5.26 ×
10-8 m (at 61 μm Ca2+). Binding
by deletion mutants of protocadherin 15 CD3 pointed to amino acids 158-179
(GenBank™ accession number {"type":"entrez-protein","attrs":{"text":"XP_238200","term_id":"293356762","term_text":"XP_238200"}}XP_238200), with homology to the comparable
region in trout hair cell protocadherin 15a-like protein, as necessary for
binding to HCN1. Amino terminus binding of HCN1 to HCN1, hypothesized to
underlie HCN1 channel formation, was also found to be
Ca2+-dependent, although the binding was skewed toward a lower
effective maximum Ca2+] than for the HCN1 interaction with
protocadherin 15 CD3. Competition may therefore exist in vivo between
the two binding sites for HCN1, with binding of HCN1 to protocadherin 15 CD3
favored between 26.5 and 68 μm Ca2+. Taken together,
the evidence supports a role for HCN1 in mechanosensory transduction of inner
ear hair cells.HCN12 is the
primary full-length HCN isoform underlying Ih
(hyperpolarization-activated, cyclic nucleotide-gated, nonselective cation
channel current) in a model hair cell preparation from the trout sacccule
(1). cAMP-gated
Ih, possibly in addition to the
mechanosensory-transduction current, sets the membrane potential for a
subpopulation of saccular hair cells
(2,
3). The membrane potential in
the saccular hair cell subpopulation is sufficiently depolarized to activate
voltage-gated calcium channels, permitting influx of calcium and secretion of
hair cell transmitter (2).
Given that saccular hair cells expressing IK1 in addition
to Ih are more hyperpolarized, not supporting activation
of the voltage-gated calcium channels, we predicted that spontaneous release
of transmitter from the subpopulation of hair cells would constitute hair
cell-generated spontaneous activity for the saccule
(1). However, little has been
previously reported on the morphological localization of the HCN1 isoform in
hair cells or possible links to structural proteins that mechanistically would
localize HCN1 in hair cells (for preliminary report, see Ref.
4). In general, little is known
about protein-protein interactions for the HCN isoforms that would modulate
Ih and/or the associated instantaneous current
(5).Protocadherin 15 is a proposed tip link protein involved in connecting
shorter stereocilia to adjacent taller stereocilia in the stereociliary array
of inner ear hair cells, facilitating the opening of the mechanosensory
transduction channel in response to auditory and vestibular stimuli. The
active tip link protein in Danio rerio is protocadherin 15a
(6), characterized by splice
variants in its carboxyl terminus. In the mammal, protocadherin 15 CD3 is
hypothesized to be a tip link protein at insertion sites in the tips of the
shorter stereocilia of the stereociliary array
(7,
8). |