Mak DO , McBride SM , Petrenko NB , Foskett JK .

GM56328 NIGMS NIH HHS , MH59937 NIMH NIH HHS, R37 GM056328 NIGMS NIH HHS , R01 MH059937 NIMH NIH HHS, R01 GM056328 NIGMS NIH HHS

	Figure 1. . Schematic diagram showing the various protocols used to isolate oocyte nuclei for nuclear patch clamp experiments.
	Figure 2. . Western blots of oocyte lysates probed with a CaM antibody (both w.t. and q.m.). Lysates from oocytes injected with CaM cRNA (w.t. or q.m.) or uninjected oocytes were used as labeled. Oocytes used for lanes A and B or C and D were from the same batches, respectively. Top arrow indicates wild-type CaM and the bottom arrow indicates the quadruple mutant CaM. The slightly faster mobility of q.m. CaM is likely a reflection of the known Ca2+-binding dependence of CaM mobility in gels (Xia et al., 1998).
	Figure 3. . Typical current traces from nuclei in NCaS bath with pipette solutions containing 10 μM InsP3. Arrows indicate closed channel current levels. (A and B) Uninjected oocytes were used. InsP3R channel activity was observed with [Ca2+]i of 755 nM (A, n = 3), whereas no channel activity was observed in a membrane patch obtained from the same region of the same nucleus with [Ca2+]i of 290 μM and the pipette solution containing 500 μM W-7 (B, n = 5). (C and D) Oocytes injected with CaM q.m. cRNA were used. InsP3R channel activity was observed with [Ca2+]i of 2.1 μM (C, n = 4), whereas no channel activity was observed in a membrane patch obtained from the same region of the same nucleus with [Ca2+]i of 290 μM (D, n = 9).
	Figure 4. . Typical current traces from nuclei in ULCaS bath isolated by protocol L. Arrows indicate closed channel current levels. Pipette solutions contained 10 μM InsP3 and [Ca2+]i as tabulated. The last current trace was obtained with −20 mV applied transmembrane potential. Other current traces were obtained with +20 mV applied potential.
	Figure 5. . [Ca2+]i dependencies of the channel Po 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 Po 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 (<τo>) 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 <τo> and higher channel Po than those in ULCaS bath, as observed. On the other hand, in [Ca2+]i > 300 nM, <τo> does not exhibit any dependence on [Ca2+]i although the mean closed channel duration (<τc>) 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 <τc>. Thus, there would be no difference between the observed Po of InsP3R in ULCaS and NCaS bath in [Ca2+]i > 300 nM, as observed.
	Figure 6. . (A and B) Typical current traces from nuclei isolated with protocol L. Arrows indicate closed channel current levels. (A) The pipette solutions contained no InsP3 and 290 μM [Ca2+]i as tabulated. (B) The pipette solutions contained 10 nM InsP3 and [Ca2+]i as tabulated. (C) [Ca2+]i dependence of the channel Po of the InsP3R in the presence of various [InsP3] as tabulated. The number of channels used to evaluate each of the data points (n) is tabulated next to the corresponding data point. Oocyte nuclei used were isolated using protocol L. The curves are simple activating Hill equation fits (Eq. 2) with the same Kact = 280 nM and Hact = 2.7. The dashed, dotted, and continuous curves have Pmax = 0.18, 0.35, and 0.84 for [InsP3] = 10 nM, 20 nM, and 10 μM, respectively.
	Figure 7. . Typical current traces obtained from nuclei isolated using different protocols, all from the same batch of oocytes. Arrows indicate closed channel current levels. All pipette solutions contained 10 μM InsP3. (A) InsP3R channel activity in 290 μM [Ca2+]i in nuclei isolated by protocol L into ULCaS, n = 5. (B) Absence of InsP3R channel activity in 290 μM [Ca2+]i in nuclei isolated by protocol LN, n = 11. (C) InsP3R channel gating in 5.5 μM [Ca2+]i (n = 2) in the same nucleus as used in B. (D) InsP3R channel activity in 290 μM [Ca2+]i in nuclei isolated by protocol LNL, n = 4.
	Figure 8. . (A) Typical current traces obtained from nuclei isolated from uninjected oocytes using protocol L with pipette solution containing 10 μM InsP3, 290 μM [Ca2+]i, and 10 μM purified CaM, n = 4. (B–D) Typical current traces obtained from nuclei isolated from oocytes expressing q.m. CaM with pipette solution containing 10 μM InsP3 and 290 μM [Ca2+]i. InsP3R channel activity was observed in 290 μM [Ca2+]i in nuclei isolated by protocol L (B, n = 6) or protocol LNL (D, n = 8), but not in nuclei isolated by protocol LN (C, n = 9). Arrows indicate closed channel current levels. (E) Histogram of InsP3R channel Po at 10 μM InsP3 and 290 μM [Ca2+]i observed in various nuclei under experimental conditions as tabulated.

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