FIGURE 12.

Gly²⁰⁶–Pro²¹⁷ deletion does not affect the association of KCa2.3 with Rab35 but decreases the association with RME-1. A, co-immunoprecipitation of KCa2.3 or ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 (Δ12) with HA-tagged Rab35 was carried out in HEK cells as described under “Experimental Procedures.” Rab35 was immunoprecipitated via an HA tag, and the subsequent blot was probed for KCa2.3. Cells expressed either Rab35 plus empty vector (1st lane), KCa2.3 plus vector (2nd lane), KCa2.3 plus HA-Rab35 (3rd and 4th lanes) or ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 plus HA-Rab35 (5th lane). The V5 Ab was used as an IgG control in 3rd lane. Lysate for each of these was probed for KCa2.3 (bottom panel) to evaluate expression. Note that ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 is expressed at a lower level than full-length KCa2.3. Similar results were observed in three separate experiments. Thus, deletion of this 12-amino acid domain does not influence the association between KCa2.3 and Rab35. B, co-immunoprecipitation of KCa2.3 or ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 (Δ12) with GFP-tagged RME-1 was carried out in HEK cells as described under the “Experimental Procedures.” RME-1 was immunoprecipitated via a GFP tag, and the subsequent blot was probed for KCa2.3. Cells expressed either KCa2.3 plus vector (1st lane), RME-1 plus empty vector (2nd lane), KCa2.3 plus GFP-RME-1 (3rd and 4th lanes), or ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 plus GFP-RME-1 (5th lane). The V5 Ab was used as an IgG control in 3rd lane. Lysate for each of these was probed for KCa2.3 (bottom panel) to evaluate expression. Note that RME-1 pulled down significantly less ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 compared with wild type KCa2.3, which cannot be accounted for by the small decrease in total ΔGly²⁰⁶–Pro²¹⁷ expression. C, cell surface expression of KCa2.3 and ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 was evaluated by biotinylation followed by streptavidin pulldown. ΔGly²⁰⁶–Pro²¹⁷ KCa2.3 was expressed at a similar level to wild type KCa2.3, indicating this cannot account for the decreased association observed upon co-IP (A).