Whole-cell currents recorded from control and transfected cells.

<p>(A & B) Representative mechanotransduction currents recorded from mouse utricle type II hair cells at P3 - P6. Bundle deflections were evoked using the protocol shown at the bottom of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008627#pone-0008627-g003" target="_blank">figure 3I</a>. Panel A shows data from a non-transfected control cell and panel B shows data from a GFP+ cell transfected with the HCN2-AYA construct. The scale bars in B apply to panels A and B. (C & D) Representative currents recorded in response to families of voltage steps that ranged between −124 mV and −64 mV in 10 mV increments. Capacitive transients and leak currents were subtracted for clarity. The scale bars in panel D apply to both panels C and D. Panel C shows I_h recorded from a non-transfected utricle type II hair cell from the same epithelium as that shown in panel A. Panel D shows a family of currents recorded from the same GFP+ shown in panel B. A family of voltage steps was used that was identical to those used to evoke the data shown in panel C. In this case, expression of the HCN2-AYA construct inhibited I_h. (E) A fluorescence image that revealed GFP expression was superimposed on a DIC image of the same field of cells. The recording pipette is visible to the right of the cell. Scale bar equals 5 µm.</p

Mechanotransduction in wild-type and HCN-deficient hair cells.

<p>Representative mechanotransduction currents evoked by hair bundle deflections. The left column of data were recorded from mouse utricle type II hair cells at P3 - P6. The data in the right column were recorded from mouse cochlear outer hair cells at P6 - P7. The scale bars at the top apply to all data in the column. The deflection protocol is shown at the bottom of trace panels I and J. Data were recorded under the following conditions: (A & B) wild-type control; (C & D) in the presence of 500 µM ZD7288; (E & F) HCN1^−/−; (G & H) HCN2^−/−; (I& J) HCN1^−/− and HCN2^−/−. Panels K-P show fluorescent images of hair cells following application of the transduction channel permeable dye, FM1-43, in utricle (P5 –P7) and organ of Corti (P6 – P9) sensory epithelia under the following conditions: (K & L) HCN1^−/−; (M & N) HCN2^−/−; (O& P) HCN1^−/− and HCN2^−/−. Uptake of FM1-43 appeared normal in all tissues examined. The scale bar in panel (O) equals 10 µm and also applies to panels K and M. The scale bar in panel (P) equals 5 µm and also applies to panels L and N. (Q) Summary of transduction currents recorded from vestibular and auditory hair cells. Maximum current amplitudes under each condition were averaged and were normalized relative to wild-type controls. Error bars show standard deviation; the number of samples is indicated above each bar.</p

Immunolocalization of HCN subunits in the inner ear.

<p>All panels show confocal images of mouse inner ear epithelia with phalloidin staining shown on the left, HCN1 in the center and the merged image on the right. Phalloidin is shown in red and HCN1 in green. All scale bars indicate 5 µm. (A) Stereociliary bundles of wild-type mouse utricle at P8 stained with an antibody directed against the N-terminus of HCN1. (B) Basolateral hair cell membranes of wild-type mouse utricle stained with the N-terminal HCN1 antibody. (C) Stereociliary bundles of wild-type mouse cochlea harvested from the apex at P8 stained with same N-terminal HCN1 antibody. (D) Confocal image of the stereociliary bundles from a P8 utricle of a HCN1^−/− mouse stained the same HCN1 antibody shown in panels A–C. (E) Wild-type utricle focused at the hair bundle level stained with a different antibody that recognizes an epitope in the C-terminus. (F) Wild-type cochlear hair bundles stained with the antibody that recognizes the epitope in the C-terminus.</p