Figure 5.

[Ca²⁺]_i dependencies of the channel P _o of the InsP₃R in oocyte nuclei isolated using various protocols (Nd, L, and LNL) and applied potentials (±20 mV) as tabulated. All pipette solutions used contained 10 μM InsP₃. The dashed curve is a simple activating Hill equation fit for the data from nuclei isolated with protocol L (large open circles). For comparison, the biphasic Hill equation fit (continuous curve) for the data points from nuclei isolated directly into NCaS bath (small filled circles) obtained in a previous study (Mak et al., 1998) are also shown. The InsP₃R channel P _o was lower in ULCaS than in NCaS at [Ca²⁺]_i ≈ 100 nM. It is possible that this reflects some intrinsic properties of the InsP₃R after exposure to the low bath [Ca²⁺]. Alternately, this may only be an artifact as a result of the movement of free Ca²⁺ ion across the open channel. With pipette [Ca²⁺]_i ≈ 100 nM, when the oocyte nucleus was in NCaS ([Ca²⁺] = 400–500 nM), the Nernst reversal potential for Ca²⁺ ions was ∼35 mV so Ca²⁺ ions moved across the open InsP₃R channel from the lumenal side to the cytoplasmic side despite an applied transmembrane voltage of 20 mV. This could cause the effective [Ca²⁺]_i at the activating Ca²⁺-binding sites on the cytoplasmic side of the channel to be higher than the free [Ca²⁺] in the bulk of the pipette solution if the Ca²⁺-binding sites are close enough to the ion conducting pore. Conversely, when the nucleus was in ULCaS ([Ca²⁺] < 5 nM), Ca²⁺ ions moved across the open InsP₃R channel in the opposite direction, down the electrical and chemical gradients, possibly lowering the effective [Ca²⁺]_i at the Ca²⁺-binding sites. In [Ca²⁺]_i < 250 nM, the mean open channel duration (<τ_o>) of the InsP₃R increases with [Ca²⁺]_i (Mak and Foskett, 1998). Therefore, if Ca²⁺ flux across the open InsP₃R channel caused the effective [Ca²⁺]_i at the activating Ca²⁺-binding sites to deviate from the free [Ca²⁺] in the bulk pipette solution, then channels in NCaS bath would have longer <τ_o> and higher channel P _o than those in ULCaS bath, as observed. On the other hand, in [Ca²⁺]_i > 300 nM, <τ_o> does not exhibit any dependence on [Ca²⁺]_i although the mean closed channel duration (<τ_c>) is still affected by [Ca²⁺]_i (Mak and Foskett, 1998). Deviation of effective [Ca²⁺] at the Ca²⁺-binding sites from the bulk free [Ca²⁺] would dissipate quickly by diffusion once the channel closed and therefore would not affect <τ_c>. Thus, there would be no difference between the observed P _o of InsP₃R in ULCaS and NCaS bath in [Ca²⁺]_i > 300 nM, as observed.