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The effects of Tmc1 Beethoven mutation on mechanotransducer channel function in cochlear hair cells.

Beurg M, Goldring AC, Fettiplace R - J. Gen. Physiol. (2015)

Bottom Line: The Ca(2+)-dependent adaptation that adjusts the operating range of the channel was also impaired in Beethoven mutants, with reduced shifts in the relationship between MT current and hair bundle displacement for adapting steps or after lowering extracellular Ca(2+); these effects may be attributed to the channel's reduced Ca(2+) permeability.Moreover, the density of stereociliary CaATPase pumps for Ca(2+) extrusion was decreased in the mutant.The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca(2+).

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Affiliation: Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706.

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Adaptive shift in different extracellular and cytoplasmic Ca2+ concentrations. (A) Superimposed MT currents in paired-pulse protocol for 1.5 mM of external Ca2+ (CaO) and 2.5 mM of internal Ca2+ (Cai). (B) Current–displacement relations for first pulse (control; closed squares) and second pulse (+step; open squares) of records in A. (C) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 0 Cai buffered with 1 mM EGTA. (D) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in C. (E) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 2.5 mM Cai. (F) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in E. Note that there were adaptive shifts in B and D, but not in F. (G) Current–voltage relationships of MT channel as in Fig. 1 G, with 1.5 mM CaO and 2.5 mM Cai (closed circles) and 0.04 mM CaO and 2.5 mM Cai (open circles). (H) Reversal potentials and permeability ratios, PCa/PCs, with initial exposure to Cao = 1.5 mM (closed circles) and prolonged prior exposure to Cao = 0.04 mM (open circles) as a function of intracellular Ca2+ concentration, Cai. Error bars represent the mean ± SD, with the number of experiments given above the points. Theoretical values are calculated from Eq. 1, assuming Cai = 0 mM (crosses). All recordings were in apical OHCs of P4–P5 mice.
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fig5: Adaptive shift in different extracellular and cytoplasmic Ca2+ concentrations. (A) Superimposed MT currents in paired-pulse protocol for 1.5 mM of external Ca2+ (CaO) and 2.5 mM of internal Ca2+ (Cai). (B) Current–displacement relations for first pulse (control; closed squares) and second pulse (+step; open squares) of records in A. (C) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 0 Cai buffered with 1 mM EGTA. (D) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in C. (E) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 2.5 mM Cai. (F) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in E. Note that there were adaptive shifts in B and D, but not in F. (G) Current–voltage relationships of MT channel as in Fig. 1 G, with 1.5 mM CaO and 2.5 mM Cai (closed circles) and 0.04 mM CaO and 2.5 mM Cai (open circles). (H) Reversal potentials and permeability ratios, PCa/PCs, with initial exposure to Cao = 1.5 mM (closed circles) and prolonged prior exposure to Cao = 0.04 mM (open circles) as a function of intracellular Ca2+ concentration, Cai. Error bars represent the mean ± SD, with the number of experiments given above the points. Theoretical values are calculated from Eq. 1, assuming Cai = 0 mM (crosses). All recordings were in apical OHCs of P4–P5 mice.

Mentions: A possible explanation for the lack of effect of high intracellular Ca2+ introduced via the patch pipette solution is that there is significant accumulation of Ca2+ at the inner face of the channel when it is open, but Ca2+ influx is still much reduced on depolarizing toward the Ca2+ equilibrium potential. Such a hypothesis is consistent with two other observations in which the two-pulse adaptive shifts in the current displacement relation were documented under different combinations of intracellular and extracellular Ca2+ (Fig. 5). First, if the hair bundle was bathed in saline containing an endolymph-like, 0.04-mM Ca2+, an adaptive shift in the current–displacement relation still occurred when recording with an intracellular solution buffered with 1 mM EGTA (Fig. 5, C and D). In low extracellular Ca2+, the efficacy of adaptation, expressed as ΔX/A, was 0.49 ± 0.11 (mean ± SD; n = 5). However, the shift was much reduced on recording with 2.5 mM of intracellular Ca2+ (Fig. 5, E and F), with a ΔX/A value of 0.11 ± 0.14 (n = 7), not significantly different from 0 (P = 0.08). Evidence that the high, 2.5 mM of intracellular Ca2+ reached the channel was that it produced partial block of the MT current: the mean amplitude of the current was 1.44 ± 0.15 nA when recording with submicromolar intracellular Ca2+ buffered with 1 mM EGTA, and was reduced to 0.61 ± 0.21 nA with 2.5 mM of intracellular Ca2+.


The effects of Tmc1 Beethoven mutation on mechanotransducer channel function in cochlear hair cells.

Beurg M, Goldring AC, Fettiplace R - J. Gen. Physiol. (2015)

Adaptive shift in different extracellular and cytoplasmic Ca2+ concentrations. (A) Superimposed MT currents in paired-pulse protocol for 1.5 mM of external Ca2+ (CaO) and 2.5 mM of internal Ca2+ (Cai). (B) Current–displacement relations for first pulse (control; closed squares) and second pulse (+step; open squares) of records in A. (C) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 0 Cai buffered with 1 mM EGTA. (D) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in C. (E) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 2.5 mM Cai. (F) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in E. Note that there were adaptive shifts in B and D, but not in F. (G) Current–voltage relationships of MT channel as in Fig. 1 G, with 1.5 mM CaO and 2.5 mM Cai (closed circles) and 0.04 mM CaO and 2.5 mM Cai (open circles). (H) Reversal potentials and permeability ratios, PCa/PCs, with initial exposure to Cao = 1.5 mM (closed circles) and prolonged prior exposure to Cao = 0.04 mM (open circles) as a function of intracellular Ca2+ concentration, Cai. Error bars represent the mean ± SD, with the number of experiments given above the points. Theoretical values are calculated from Eq. 1, assuming Cai = 0 mM (crosses). All recordings were in apical OHCs of P4–P5 mice.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Adaptive shift in different extracellular and cytoplasmic Ca2+ concentrations. (A) Superimposed MT currents in paired-pulse protocol for 1.5 mM of external Ca2+ (CaO) and 2.5 mM of internal Ca2+ (Cai). (B) Current–displacement relations for first pulse (control; closed squares) and second pulse (+step; open squares) of records in A. (C) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 0 Cai buffered with 1 mM EGTA. (D) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in C. (E) Superimposed MT currents in paired-pulse protocol for 0.04 mM CaO and 2.5 mM Cai. (F) Current–displacement relations for first pulse (closed squares) and second pulse (open squares) for records in E. Note that there were adaptive shifts in B and D, but not in F. (G) Current–voltage relationships of MT channel as in Fig. 1 G, with 1.5 mM CaO and 2.5 mM Cai (closed circles) and 0.04 mM CaO and 2.5 mM Cai (open circles). (H) Reversal potentials and permeability ratios, PCa/PCs, with initial exposure to Cao = 1.5 mM (closed circles) and prolonged prior exposure to Cao = 0.04 mM (open circles) as a function of intracellular Ca2+ concentration, Cai. Error bars represent the mean ± SD, with the number of experiments given above the points. Theoretical values are calculated from Eq. 1, assuming Cai = 0 mM (crosses). All recordings were in apical OHCs of P4–P5 mice.
Mentions: A possible explanation for the lack of effect of high intracellular Ca2+ introduced via the patch pipette solution is that there is significant accumulation of Ca2+ at the inner face of the channel when it is open, but Ca2+ influx is still much reduced on depolarizing toward the Ca2+ equilibrium potential. Such a hypothesis is consistent with two other observations in which the two-pulse adaptive shifts in the current displacement relation were documented under different combinations of intracellular and extracellular Ca2+ (Fig. 5). First, if the hair bundle was bathed in saline containing an endolymph-like, 0.04-mM Ca2+, an adaptive shift in the current–displacement relation still occurred when recording with an intracellular solution buffered with 1 mM EGTA (Fig. 5, C and D). In low extracellular Ca2+, the efficacy of adaptation, expressed as ΔX/A, was 0.49 ± 0.11 (mean ± SD; n = 5). However, the shift was much reduced on recording with 2.5 mM of intracellular Ca2+ (Fig. 5, E and F), with a ΔX/A value of 0.11 ± 0.14 (n = 7), not significantly different from 0 (P = 0.08). Evidence that the high, 2.5 mM of intracellular Ca2+ reached the channel was that it produced partial block of the MT current: the mean amplitude of the current was 1.44 ± 0.15 nA when recording with submicromolar intracellular Ca2+ buffered with 1 mM EGTA, and was reduced to 0.61 ± 0.21 nA with 2.5 mM of intracellular Ca2+.

Bottom Line: The Ca(2+)-dependent adaptation that adjusts the operating range of the channel was also impaired in Beethoven mutants, with reduced shifts in the relationship between MT current and hair bundle displacement for adapting steps or after lowering extracellular Ca(2+); these effects may be attributed to the channel's reduced Ca(2+) permeability.Moreover, the density of stereociliary CaATPase pumps for Ca(2+) extrusion was decreased in the mutant.The results suggest that a major component of channel adaptation is regulated by changes in intracellular Ca(2+).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroscience, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706.

Show MeSH
Related in: MedlinePlus