The regulatory effects of intracellular Ca^^ on T4 OHC currents were investigated using the photolabile Ca^^ chelator DM-Nitrophen. Whole-cell currents recorded before and during Ca^"" release from this chelator were compared. Currents were recorded at -110 mV and at 0 mV. The former
voltage was chosen to investigate the effects of increased intracellular Ca^^ on Ik alone. The latter voltage was chosen to allow the Ca^"" sensitivity of Iki4 to be
investigated without contamination by a non-selective cation current, observed in isolated OHCs (Housley and Ashmore, 1992). The maximum response of the cells to the photolysis of DM-Nitrophen was compared to the pre-photolysis level.
T4 outer row OHC currents can be modulated by raised intracellular Ca^^ levels (Figure 3.15a). The maximum effect was reached ~3 seconds after the start of the UV exposure. Raised intracellular Ca^^ concentrations resulted in seven of the nine cells tested showing a very slight reduction of their outward current at the holding potential of -60 mV (0.025 ± 0.008 nA). The other two cells showed no change in their current at this voltage. In all the cells recorded, outward currents at 0 mV increased in amplitude by 0.17 ± 0.12 nA (n=9). No significant difference was observed in the time constant of current onset at 0 mV between control and test traces (P = 0.15, test f. Table 1, Appendix 1). Only one of the nine cells tested showed a change in the current level at -110 mV. For this one cell, the increase in current was very small, being just 0.02 nA. The effects of photolysis were fully reversible within 2 minutes.
To attempt to correlate the observed Ca^^ sensitivity with one of the currents described above, the experiments were repeated in the presence of millimolar concentrations of 4AP. 4AP blocks the outward current activated above -40 mV and may therefore help to discriminate between the Ca^^ effects on the currents activated above and below -40 mV. In the presence of 4AP, the responses of the cells were variable. Three out of six cells showed an increase in the inward current at the holding potential of -60 mV of 0.093 + 0.049 nA. The other three cells showed no change in the holding current. Four of the six cells tested showed a response to raised intracellular Ca^"" at 0 mV of 0.221 ± 0.15 nA. Five showed an increase in their inward current of 0.123 ± 0.098 nA. At 0 mV, current activation did get faster with raised intracellular (Figure 3.15b). However, this change in the time constant of current onset was unmeasurable due to the contamination of the current by the motor transient. The highly variable responses observed in this set of experiments may reflect:
a) the different loading of chelator between different cells
b) the increased age of the cells by the time intracellular Ca^^ was increased compared to control cells due to the time required to bath perfuse the cells with 4AP.
The effects of photolysis were not always fully reversible. 32% of all the cells investigated exhibited some permenant change following photolysis.
These results indicate that lkT4 is unlikely to be Ca^^ sensitive as the
Ca^^ responses of the OHCs are maintained in the presence of millimolar
concentrations of 4AP. Ca^^ could be activating the TEA-sensitive conductance. However, this conductance must play a very small part in the Ca^^ response, as significant increases in current with the photolysis of DM-Nitrophen were still observed in the presence of TEA (data not shown). The increase in the inward current at -60 mV is not consistent with the activation of a conductance. Thus, it seems unlikely that Ik.n, the potassium conductance activated around the zero-current potential of the cell, would contribute towards this response.
However, the Ca^^ sensitivity of this current cannot be ruled out as a decrease in the extracellular Ca^^ concentration of the perfusate did, in one experiment, cause a decrease in the conductance of the cell both above and below -50 mV,
+ [Ca^^i release a 0.5 nA 50 ms
>
b =
+ [Ca2+]j release 0 mV -110 mV -60 mVFigure 3.15 Effects of increasing intracellular Ca^* by photolysis of DM-Nitrophen on T4 OHC currents, a, control currents recorded before and during photolysis, b, currents recorded before and during photolysis in the presence of 3 mM 4AP. Voltage protocol shown in the bottom panel.
a result also observed by Raybould and Housley (1997). Additionally, dagger and Ashmore (1999) have shown the current activated around the zero-current of the cell to be sensitive. Finally, these results could be explained by the activation of a cation conductance which, as well as increasing the inward and outward conductances, would also increase the inward current at the holding potential of the cell. Although the aim of recording the current at 0 mV was to minimize the effect of such a current, voltage errors due to uncompensated series resistance would keep the voltage just below 0 mV, allowing the effect of raised intracellular Ca^^ on this current to be observed . It is unlikely that the cation conductance in question is associated with the transducer current as Ca^^ responses could also be obtained from OHCs in the presence of 100 p-M DH- streptomycin. However, a Ca^^ sensitive cation conductance, associated with the basolateral membrane of the hair cell, has been observed in isolated preparations (Housley and Ashmore, 1992, Van den Abbeele, Tran Ba Huy, Teulon, 1994). The amount of current carried through the channels associated
with this conductance increases with time after isolation and time in the whole cell configuration possibly due to intracellular Ca^^ accumulation. The latter effect could certainly explain why increased and more variable responses were observed in the presence of 4AP, as these cells had been patched longer before photolysis to allow for the bath perfusion of 4AP. However, in a freshly excised cochlea, a cation conductance was not obvious (Figure 3.5) which may indicate that the channel carrying this cation conductance is expressed, but not functioning in T4 OHCs unless the concentration of intracellular Ca^^ is
increased.
It seems highly unlikely that these results can be exclusively explained by the Ca^‘" activation of a cation conductance when there is also evidence for Ca^^ activation of lk,n (Housley and Ashmore, 1992; dagger and Ashmore, 1999; Raybould and Housley, 1997). Therefore, the story is probably far more
complex than the explanation given above and most likely involves the
combined activation of lk,n and the cation conductance, the observed changes being the net effect of the two currents.
3.4
Discussion
This preparation provides an ideal means of examining the physiological characteristics of OHC for two reasons:
a) the significantly more negative zero-current potential of in situ OHCs compared to in vitro OHCs, that tends towards the zero-current
potential for in vivo OHCs indicates that this preparation provides an ideal physiological environment in which to study OHCs without the complications of an in vivo preparation.
b) it allows the recording site along the basilar membrane to be unequivocally identified and thus, the electrophysiological
characteristics of OHC to be closely correlated with the best tuning frequency for that point along the cochlear length
The identity of the ion channels carrying the basolateral membrane currents of mammalian T4 OHCs has been examined by characterizing the
currents according their kinetics, pharmacology and Ca^^ sensitivity. The results are summarized in Figure 3.16. These results are grossly similar to previous studies, in so far as they demonstrate that in situ OHCs exhibit a negative zero- current potential and express at least two K^-dependent membrane currents, one active around the zero-current potential of the cell, the other above -40 mV. However, these results extend previous observations by suggesting that there are at least two components to each of these previously identified
conductances. These conclusions help to bring together the results from other studies.
3.4.1 B a s o la te ra l m e m b ra n e c o n d u c ta n c e s a c tiv a te d a b o v e