Fig. 2.

SPAK2 is a strong inhibitor of NKCC1 but a relatively weak inhibitor of NKCC2. A–D: data are expressed relative to uptake by NKCC2-injected oocytes, set to 100% for each condition ± SE; n = 4–5 (different frogs). Uptake by water-injected oocytes was only 3–17% of that of oocytes injected with NKCC2 alone; bumetanide inhibited uptake by oocytes injected with NKCC2 alone by 90–96% (data not shown), confirming that the majority of uptake is through injected NKCC2 (A) At least 30 oocytes were injected as indicated with 20 ng of NKCC2 and 10 ng of KS-SPAK or SPAK2 cRNAs used. In this experiment, the preincubation and uptake conditions differ from those used in Fig. 1 (see materials and methods). The night before the uptake experiment, each injection group was divided into groups of 10 and preincubated in the appropriate solution. ⁸⁶Rb uptake was then measured for the pooled groups of oocytes in the appropriate uptake medium. *P ≤ 0.01, lower activity, and #P < 0.01, higher activity, compared with the NKCC2-injected group at the same tonicity. B: 10–20 oocytes were injected as indicated with 20 ng of NKCC1 or NKCC2 cRNA and 10 ng of KS-SPAK or SPAK2 cRNA. ⁸⁶Rb uptake was performed after 3 days under hypotonic conditions, following an overnight preincubation in hypotonic low Cl⁻ solution. *P ≤ 0.001, lower degree of inhibition of uptake by SPAK2 in NKCC2- than in NKCC1-coinjected oocytes. C: 12–20 oocytes were injected with 20 ng of NKCC2 cRNA and KS-SPAK and/or SPAK2 cRNA (in ng) as indicated. ⁸⁶Rb uptake was performed after 3 days under hypertonic conditions, following an overnight preincubation in hypertonic solution. *P < 0.01, lower uptake compared with oocytes injected with NKCC2 only; #P < 0.05, higher uptake by oocytes injected with NKCC2, KS-SPAK and SPAK2 (20 ng), compared with water-injected oocytes. D: 10–22 oocytes were injected with 20 ng of NKCC2 cRNA and constitutively active FL-SPAK (CA-SPAK), KS-SPAK and/or SPAK2 cRNA (in ng) as indicated. ⁸⁶Rb uptake was performed after 3 days under hypotonic conditions, following an overnight preincubation in hypotonic low Cl⁻ solution. *P < 0.001, lower uptake compared with oocytes injected with NKCC2 only; #P < 0.05, lower uptake by oocytes injected with NKCC2 and CA-SPAK with either KS-SPAK (10 ng) or SPAK2 (20 ng), compared with oocytes injected with NKCC2 and CA-SPAK only. C and D, bottom: representative Western blots confirming expression of SPAK isoforms.