The amino terminus of Slob, Slowpoke channel binding protein, critically influences its modulation of the channel

Abstract

The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance-voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance-voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

Figure 1.

Six Slob proteins from a single Slob gene. There are at least four different transcripts of the slob gene in vivo. These transcripts can initiate translations at either Met1 (M1) or Met124 (M124), and contain or exclude the spliceable exon 7 (green). Therefore, at least four different Slob proteins can be produced. We named them Slob71, Slob65, Slob57, and Slob51 based on their molecular weights. A third Met (M163) can be recognized by the ribosome to translate two more Slobs: Slob53 and Slob47. The epitope-tagged Slob used in our previous study was Slob57, with an amino-terminal HA epitope appended to Arg151 (R151). All Slobs contain the dSlo binding region (brown). All amino acid numbering is relative to the first Met (M1) in Slob71.

Figure 2.

Heterologous expression of Slobs. (A) Different Slobs were transiently transfected into CHO cells. On the second day, proteins in cell lysates were separated by gel electrophoresis, transferred onto nitrocellulose membranes, and blotted with anti-Slob antibody. Lane 1, Slob71 construct with M124L mutation; lane 2, Slob65 construct with M124L mutation; lane 3, Slob57 construct; lane 4, Slob51 construct; lane 5, pIRES2-EGFP vector only. (B) Individual Slob variants can be expressed by mutating one or more translational start sites (see Fig. 1). Lane 1, Slob71 construct with M124L and M163L mutations; lane 2, Slob65 construct with M124L and M163L mutations; lane 3, Slob57 construct with M163L mutation; lane 4, Slob57 construct with M124L mutation, which expresses only Slob53; lane 5, Slob51 construct with M163L mutation; lane 6, Slob51 construct with M124L mutation, which expresses only Slob47; lane 7, pIRES2-EGFP vector only.

Figure 3.

Modulation of dSlo by Slob71, Slob65, Slob53, and Slob47. Whole cell currents evoked by depolarizing voltage steps (G) with 110 μM free Ca²⁺, in cells transfected with vector alone (A), dSlo alone (B), or together with different Slobs as indicated (C–F). Currents were elicited by a 350-ms test pulse to different voltages from a holding potential of −80 mV, followed by hyperpolarization to −120 mV to measure tail currents.

Figure 4.

Conductance–voltage relationship for dSlo. The conductance–voltage relationships for dSlo transfected alone or together with Slob71, Slob65, Slob53, or Slob47 were measured from tail currents with 110 μM free Ca²⁺, as shown in Fig. 3. The conductance–voltage relationship was generated from peak tail currents as described in materials and methods.

Figure 5.

Modulation of dSlo by Slob57 and Slob51. Whole cell currents, with 110 μM free Ca²⁺, were evoked by either 350 ms (A–C) or 100 ms (D–F) depolarizing pulses, followed by hyperpolarization to −120 mV to measure tail currents. dSlo was expressed either alone (A and D) or together with either Slob57 (B and E) or Slob51 (C and F).

Figure 6.

Short pulse conductance–voltage relationship for dSlo. Same as Fig. 4, except the effects of Slob57 and Slob51 were measured using the shorter pulse protocol in Fig. 5 (D–F).

Figure 7.

Slob57 and Slob51 change the deactivation rate of dSlo. Deactivating tail currents following a 100-ms test pulse to +40 mV, with 110 μM free Ca²⁺, in the absence or presence of Slob57 or Slob51, were normalized and superimposed. The deactivation time constants for dSlo expressed alone, or together with Slob57 or Slob51, were obtained by fitting the tail currents with exponential functions. Both the current traces themselves and the exponential fits to the data (smooth lines) are shown in the figure. When dSlo is expressed without Slobs, in the voltage range from −20 to +40 mV, the tail currents can be fitted with a single exponential function with a mean, voltage-independent τ of 8.4 ± 0.5 ms. In the presence of Slob57 or Slob51, the tail currents can only be fitted, in the voltage range from +20 to +180 mV, with two voltage-independent exponential functions. Both the τ_f and the τ_s of dSlo in the presence of Slob57 or Slob51 are significantly different (P < 0.001) from the τ of dSlo expressed alone.

Figure 8.

Conductance–voltage relationship for dSlo with lower concentrations of free Ca²⁺. Same as Fig. 4, except the effects of Slob53 and Slob71 were measured with 20 μM (filled symbols) or 40 μM (open symbols) free Ca²⁺.

Figure 9.

The apparent inactivation of dSlo caused by Slob57 is Ca²⁺ independent. Whole cell currents, with (A) Ca²⁺-free solution (no added Ca²⁺), (B) 4 μM free Ca²⁺, (C) 20 μM free Ca²⁺, (D) 40 μM free Ca²⁺, were evoked by the same pulse protocol as shown in Fig. 3 G. dSlo was expressed either alone or together with Slob57 as indicated.