passages: PMC-2151581-sec-12__text
passages: sec
passages: Ca2+ Dependence of Slob57 Modulation of the Voltage Dependence of Activation of dSlo
  Ca2+ plays a fundamental role in regulating the dSlo response to membrane depolarization. One of the three known Ca2+ binding sites (Xia et al., 2002; Bao et al., 2002; Zeng et al., 2005b), known as the calcium bowl (Schreiber and Salkoff, 1997), contributes to the high-affinity Ca2+ sensitivity of the channel (Schreiber and Salkoff, 1997; Schreiber et al., 1999; Braun and Sy, 2001; Bao et al., 2002; Niu and Magleby, 2002; Xia et al., 2002; Bao et al., 2004; Zeng et al., 2005b). We used a mutated dSlo channel with substitution of Asp966-Asp970 by Asn966-Asn970 within its calcium bowl, which greatly reduces the channel's calcium sensitivity (Bian et al., 2001), to determine whether Ca2+ participates in Slob57 modulation of dSlo. This mutated channel, dSloD5N5, still binds to full-length and truncated Slobs (Fig. 1 B, lanes 4-6). During a 350-ms test pulse (Fig. 7 E), Slob57 causes dSloD5N5 inactivation (Fig. 7 B), consistent with our previous finding that the inactivation caused by Slob57 is Ca2+ independent (Zeng et al., 2005a). After deletion of the amino-terminal residues 2-6 of Slob57, the inactivation of dSloD5N5 is eliminated (Fig. 7 C), and further truncation of Slob57 to Arg16 also eliminates the inactivation (Fig. 7 D), just as observed with wild-type dSlo (Fig. 2, C and D).
  Figure 7.
  Slob57 causes inactivation of dSloD5N5. Same as Fig. 2, except that the mutated channel dSloD5N5 was used instead of wild-type dSlo. (A) dSloD5N5 alone, (B) dSloD5N5 together with Slob57, (C) dSloD5N5 together with Slob57DeltaN5, (D) dSloD5N5 together with Slob57DeltaN15, (E) pulse protocol.
  We next used a 100-ms test pulse (during which no inactivation occurs) to measure the voltage dependence of activation of dSloD5N5 in the presence of 110 muM free Ca2+, as only at high concentrations of free Ca2+ can tail current saturation be achieved with the mutant channel (Fig. 8). As shown in Fig. 8, Slob57 does not rightward shift the conductance-voltage relationship of dSloD5N5. Because dSloD5N5 can still respond to an increment of free Ca2+ at high concentration (Bian et al., 2001), we then increased the concentration of free Ca2+ to 300 muM. As shown in Fig. 8, the conductance-voltage relationship of dSloD5N5 shifts to less depolarized voltages when the concentration of free Ca2+ is increased from 110 to 300 muM, but again the V1/2 is not modulated by Slob57.
  Figure 8.
  Conductance-voltage relationships for dSloD5N5. Same protocol as Fig. 6, except that the mutated channel dSloD5N5 was used in the presence (circles) or absence (squares) of Slob57, and the concentration of free Ca2+ was either 110 muM (filled symbols) or 300 muM (open symbols).
  We next measured the voltage dependence of activation of wild-type dSlo, in the presence or absence of Slob57, at free Ca2+ concentrations ranging from 20 to 300 muM. As shown in Fig. 9 A (and by many previous investigators), in the absence of Slob57, the voltage-conductance relationship of dSlo shifts to less depolarized voltages as the free Ca2+ concentration is increased in this range. The V1/2 decreases from 85 to -22 mV, a change of 107 mV, when the concentration of free Ca2+ is increased from 20 to 300 muM. In contrast, when dSlo is coexpressed with Slob57, this increment of free Ca2+ concentration shifts the V1/2 to a much lesser extent (Fig. 9 B and Fig. 10 A). Note also that the effect of Slob57 on the V1/2 is more apparent at higher free Ca2+ concentrations (Fig. 10 A). To illustrate this better, we plotted the V1/2 change evoked by Slob57 (DeltaV1/2) as a function of the free Ca2+ concentration (Fig. 10 B) to demonstrate that the magnitude of the modulatory shift in V1/2 evoked by Slob57 itself is calcium dependent.
  Figure 9.
  Effect of calcium on dSlo voltage dependence of activation in the absence or presence of Slob57. Same as Fig. 6, except that the measurements of dSlo expressed alone (A) or together with Slob57 (B) were performed in the presence of different concentrations of free Ca2+: 20 muM (diamonds), 40 muM (inverted triangles), 80 muM (triangles), 110 muM (squares), and 300 muM (circles).
  Figure 10.
  Interaction between Slob57 and calcium. The V1/2 (A) of the conductance-voltage relationship for dSlo expressed alone (O) or together with Slob57 (*), taken from Fig. 9, and the difference in V1/2 (DeltaV1/2) evoked by Slob57 (B) are plotted as a function of the concentration of free Ca2+.
  