The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.auditory system ͉ cochlea ͉ deafness ͉ knockout mice ͉ sialyltransferase G angliosides [i.e., glycosphingolipids (GSLs)] containing sialic acid are abundant in central nervous tissues and are considered to have important roles in controlling development, proliferation, differentiation, and maintenance of neural tissues and cells (1). Gangliosides usually reside in the outer leaflet of the cell membrane and are concentrated in microdomains specialized for cell adhesion and cell signaling (2, 3). The first product in the biosynthetic pathway of the ganglio-series gangliosides is GM3, which serves as a common precursor for the a-series and b-series gangliosides (Fig. 1). The sialyltransferase responsible for GM3 synthesis is the GM3 synthase (EC 2.4.99.9), also known by the names SAT-I (used here), ST3GalV, and Siat 9. The gene encoding this enzyme has been identified by several research laboratories, including ours (4-6).In the study presented here, we generated mice deficient in GM3 synthase (SAT-I Ϫ/Ϫ mice) (7) and examined their general behavior, including their motor function, learning and memory, and sensory function. Interestingly, SAT-I Ϫ/Ϫ mice exhibited no startle reflex in response to various acoustic stimulations, yet they did demonstrate normal startle responses to air puffing, suggesting a hearing impairment. Electrophysiological and histological analyses of the auditory system of the SAT-I Ϫ/Ϫ mice further revealed that the organ of Corti in the inner ear is selectively degenerated in these animals. ResultsAbsence of Acoustic Startle Response in SAT-I Null Mice. GM3 synthase null (SAT...

1. Electrical and pharmacologic properties of ATP-induced current in outer hair cells isolated from guinea pig cochlea were investigated in the whole-cell recording mode by the use of a conventional patch-clamp technique. 2. Under current-clamp conditions, rapid application of ATP depolarized the outer hair cells resulting in an increase in conductance. The ATP-induced response did not show any desensitization during a continuous application. 3. At a holding potential of -70 mV, the ATP-induced inward current increased in a sigmoidal fashion over the concentration range between 3 microM and 1 mM. The half-maximum concentration (EC50) was 12 microM and the Hill coefficient was 0.93. 4. The ATP-induced current had a reversal potential near 6 mV, which was close to the theoretical value (1 mV) calculated from the Goldman-Hodgkin-Katz equation for permeable intra- and extracellular cations. 5. In the current-voltage (I-V) relationship for the ATP response, a slight inward-going rectification was observed at more positive potentials than the reversal potential. 6. The substitution of extracellular Na+ by equimolar choline+ shifted the reversal potential of the ATP-induced current to more negative values. The substitution of Cs+ in the internal solution by N-methyl-D-glucamine+ (NMG+) shifted it in the positive direction. The reversal potential of ATP-induced current was also shifted to positive values with increasing extracellular Ca2+ concentration. A decrease of intracellular Cl- by gluconate- did not affect the reversal potential, thereby indicating that the ATP-induced current is carried through a large cation channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+ channel kinetics were studied by recording single-channel currents in the cell-attached patch configuration of the patch-clamp technique in single ventricular cells isolated from guinea pig hearts. The inactivation time course of ensemble currents was accelerated, and the peak amplitude increased temporarily and then decreased within a few minutes after the gigaohm seal formation. After reaching a new steady state, the inactivation-voltage relation was found to have shifted to more negative potentials. The potential of half-maximal inactivation was more negative by 20-31 mV from the resting potential or between -96 and -112 mV. The voltage dependency of the channel activation also shifted. Although the cell membrane was depolarized using the whole cell patch-clamp electrode and single-channel currents were recorded with an independent cell-attached electrode, the shift of the inactivation curve was also evident. Complete removal of Ca2+ using 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution failed to prevent the shift. Increasing Ca2+ to 10 mM, however, reduced magnitude of the shift significantly. Involvement of an increased membrane fluidity and surface potential of the glass pipette to the shift is discussed.

49 mV, while the inward current was not blocked.5. The Na+-activated K+ channel showed frequent subconductance levels. The variance-mean analysis resolved at least ten major sublevels. The density distribution of the sublevels were measured by composing the conventional amplitude histogram, excluding clear closed state currents, and then dividing the histogram into five segments. The probability of staying in each segment (P.) was almost always voltage independent, and the grand averages were P1 = 9.5 + 5.9 %, P2 = 6-3± 2-1 %, P3 = 4-2 +1P8 %, P4 = 7-8 + 2-5 %, and P5 = 39-3 +5-6 %, from the lowest segment, respectively.6. The values of Pn in partially blocked conditions by Na+ and Mg2+ (outward current) were not clearly different from those without any channel block (inward current). The values of Pn, measured before and after applying Ba2+ in the pipette, were also very similar.7. The above findings indicate that the inward-going rectification of the Na+-activated K+ channel is due to the Na+ and Mg2+ block. The subconductance of the channel is not due to any channel block by Na+ or Mg2+, but may be attributable to multiple open states of a single-barrel channel, which has a large conductance. The channel may be blocked from any open conformation with an equal probability and with very fast kinetics.

Two-staged intact canal wall tympanoplasty is a common operation for treatment of middle ear cholesteatoma. MRI provides better tissue differentiation of the middle ear and/or mastoid, which often become occupied with soft density tissue after the first operation. If MRI was able to detect the presence of a recurrent or residual cholesteatoma with sufficient sensitivity and specificity, this may facilitate a decrease in the number of second-look procedures. This study compared MRI findings to surgical findings at second-look surgery and calculated the correlation rates between the two sets of findings. Thirty ears having undergone intact canal wall tympanoplasty for cholesteatoma at the initial operation were examined by MRI prior to the second look. Otoscopic findings of the tympanic membrane were nonsuspect in all cases. The true positive rate was 11/30 (37%) and the true negative rate was 10/30 (33%), leading to a radiosurgical correlation of 70%, whereas the false positive rate was 6/30 (20%) and the false negative rate was 3/30 (10%). This indicates that 30% of the MRI findings were incorrect. Therefore, at the present time, MRI does not appear as a likely replacement for second-look surgery in cases of intact canal wall tympanoplasty.