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	Figure 1. Modulators of PKC determine KCNKØ current magnitude. Macroscopic KCNKØ channels currents measured by two-electrode voltage clamp in 20 mM potassium solution with or without 50 nM PMA or 2 μM staurosporine (see materials and methods). (A) Currents measured during the application of PMA and then staurosporine; the oocyte was held at −40 mV and stepped to 25 mV for 250 ms with a 20-s interpulse interval. The response to PMA varied among batches of oocytes from 3–11-fold; this appeared to reflect different levels of prior activation as a consistent 10 ± 1-fold increase in currents was observed when PMA and staurosporine treatments were compared (mean ± SEM, n = 10). Zero current is indicated. (B) Raw current traces for the oocyte in A at various times as follows: (1) control solution; (2) after 20 min in PMA; and (3) after 20 min in staurosporine. The cell was held at −80 mV and studied at these times by 250-ms steps from−150 to 60 mV in 30-mV increments, and then studied for 75 ms at −150 mV with a 2-s interpulse interval. (C) Currents measured at 25 mV by the protocol in A during 10-min activation by 50 nM PMA, 6-min wash with control solution, 2-min inhibition by 4 μM bisindolylmaleimide I, and then a 20-min reactivation by 50 nM PMA. Zero current is indicated.
	Figure 2. Regulation of single KCNKØ channels. Single KCNKØ channels were studied in on-cell patches held at 60 mV with 140 mM potassium solution in the absence or presence of PMA or staurosporine (see materials and methods). Open (O) and closed (C) state levels are indicated. Unitary current amplitudes of wild-type KCNKØ channels studied in control, PMA, and staurosporine solutions were indistinguishable (3.23 ± 0.04, 3.22 ± 0.07, and 3.24 ± 0.06 pA, respectively, mean ± SEM, filtered at 2 kHz, n = 3). (A) Single KCNKØ channel in control solution filtered at 20 Hz. (Inset) Expanded trace from the indicated region filtered at 2 kHz. (B) Single KCNKØ channel after 20-min incubation with 50 nM PMA filtered at 20 Hz. (C) Single KCNKØ channel after 20-min incubation with 2 μM staurosporine filtered at 20 Hz.
	Figure 3. Regulation alters occupancy of the long-lived closed state. Single KCNKØ channels were studied as in Fig. 2. (A) Open time histogram obtained from 17 s of single KCNKØ channel recordings filtered at 3 kHz under control conditions. (B) Closed time histograms under control conditions; (left) obtained from 17 s of single KCNKØ channel recordings filtered at 3 kHz; (right) obtained from 41 min of recordings of single KCNKØ channels filtered at 100 Hz. Four channel records were combined for this analysis. (C) Open time histogram obtained from 22 s of single KCNKØ channel recordings filtered at 3 kHz in the presence of PMA. (D) Closed time histograms in the presence of PMA: (left) obtained from 22 s of single KCNKØ channel recordings filtered at 3 kHz; and (right) obtained from 54 min of recordings of single KCNKØ channels filtered at 100 Hz. Four channel records were combined for this analysis. (E) Four KCNKØ channels were recorded in an on-cell patch at 60 mV with PMA in the bath and then in staurosporine as indicated. Data were sampled at 940 Hz and filtered at 20 Hz.
	Figure 4. Truncated (KCNKΔ299-1001) channels are not subject to regulation. Macroscopic KCNKØ currents were measured as in Fig. 1. (A) Predicted membrane topology of KCNKØ indicating two P loop domains (P1 and P2), four transmembrane segments and the ∼700-residue segment that is deleted in KCNKΔ299-1001 subunits (boxed). (B) Raw current traces for an oocyte expressing KCNKΔ299-1001 channels under various conditions by the protocol in Fig. 1 B: (1) control solution; (2) after 10-min perfusion with PMA; and (3) after 10-min perfusion with staurosporine. (C) Currents measured during application of control, PMA, and staurosporine solutions. The oocyte was held at −40 mV and stepped to 25 mV for 250 ms with a 20-s interpulse interval. Data for B was collected at the indicated times. (D) Normalized mean currents measured in groups of 17 oocytes at 25 mV after 20- min incubation in the following: (C) 20 mM potassium solution (open bars); (S) 2 μM staurosporine (gray bars); or (P) 50 nM PMA (black bars). Bars are mean ± SEM for current in experimental compared with control solution.
	Figure 5. Unitary conductance of wild-type and KCNKΔ299-1001 channels is the same. Single channels were studied during open bursts in on-cell patches with 140 mM KCl solution at the indicated voltages. Open (O) and closed (C) state levels are indicated. Data were filtered at 2 kHz and sampled at 20 kHz. (A) Sample recording of a single wild-type KCNKØ channel at four indicated voltages. (B) Sample recording of a single KCNKΔ299-1001 channel as in A. (C) Current–voltage relationships of the channels in A (•) and B (○). The line is a linear fit to the data; slope conductances were indistinguishable (see results for values).
	Figure 6. Closed and open states of KCNKΔ299-1001 channels are similar to wild type. Single KCNKΔ299-1001 channels were studied as in Fig. 2 and Fig. 3. Open (O) and closed (C) state levels are indicated. (A) Sample 5-min recording of a single channel at 60 mV filtered at 20 Hz. (B) Expansion of the indicated portion of A, filtered at 3 kHz, showing interburst behavior. (C) Open time histogram from 25 s of recording during open bursts filtered at 3 kHz. (D) Closed time histograms: (left) obtained from 25 s of recording during open bursts filtered at 3 kHz; and (right) obtained from 23 min of single KCNKØ channel recording filtered at 100 Hz. Five single channel records were combined for this analysis.
	Figure 7. Multiple regulatory pathways act via the KCNKØ carboxy terminus. Macroscopic KCNKØ currents measured as in Fig. 1 after equilibration for 10 min in the initial bath solution with scale bars that represent 5 min and 0.5 μA. Single channels were studied as in Fig. 2. (A) Macroscopic currents measured in the constant presence of the PKC inhibitor bisindolylmaleimide I (4 μM) during application of 50 nM PMA and then a combination of IBMX (1 mM) and forskolin (20 μM) to activate PKA. The average increase in current under those conditions was 4.5 ± 1 (n = 5). (B) Macroscopic currents measured in the constant presence of the PKC inhibitor bisindolylmaleimide I (4 μM) during the application of 50 nM PMA and then microinjection of 8-Br-cGMP (9.2 nl of 30 mM solution) to activate PKG. The average increase in current under those conditions was 4.4 ± 1 (n = 5). (C) Macroscopic currents measured in the constant presence of the PKA inhibitor H89 (5 μM) during application of a combination of IBMX (1 mM) and forskolin (20 μM) and then 50 nM PMA to activate PKC. The average increase in current under those conditions was 6 ± 1 (n = 5). (D) Macroscopic currents measured during microinjection of 8-Br-cGMP (9.2 nl of 30 mM solution) to activate PKG, followed by application of IBMX (1 mM) and forskolin (20 μM) to activate PKA and 50 nM PMA to activate PKC. The average increase in current under those conditions was 9.5 ± 0.8 (n = 5). (E) Sample single channel trace at 60 mV (filtered at 20 Hz) during concurrent exposure to the PKC inhibitor bisindolylmaleimide I (4 μM) and of IBMX (1 mM) and forskolin (20 μM) to activate PKA. Open (O) and closed (C) state levels are indicated. (F) Open time histogram for single KCNKØ channels exposed concurrently to a PKC inhibitor and PKA activators as in E, as determined from 18 s of single channel recording filtered at 3 kHz. (G) Closed time histograms for single KCNKØ channels exposed concurrently to a PKC inhibitor and PKA activators as in E: (left) determined from 18 s of single channel recording, filtered at 3 kHz; and (right) determined from 45 min of single channel recording, filtered at 50 Hz.
	Figure 8. KCNKØ residues that mediate PKC and PKA effects are distinct. Macroscopic currents measured as in Fig. 1; single channels were studied as in Fig. 2. (A) Each of the 11 consensus sites for PKC-mediated phosphorylation was mutated individually to a nonpolar residue. Normalized mean currents were measured in groups of eight oocytes expressing the indicated channel after 20-min incubation with 50 nM PMA. Bars are mean ± SEM for current in experimental compared with staurosporine solution. (B) Normalized mean current in groups of 8–10 oocytes expressing the indicated KCNKØ channel type during application of 50 nM PMA; the currents in each cell were normalized to untreated current level before averaging. (C) After truncation at various sites, the normalized mean currents were measured in groups of 5–10 oocytes expressing the indicated channel after 5 min exposure to 50 nM PMA. Bars represent mean ± SEM for the ratio of current at 5 min to initial current. (D) Sample trace for a single KCNKØ-S270LΔ872-1001 channel at 60 mV (filtered at 20 Hz) under control conditions and in the presence of 50 nM PMA. Open (O) and closed (C) levels are indicated. (E) Closed time histograms for single KCNKØ-S270LΔ872 channels: (left) determined from 18 s of single channel open burst recording filtered at 3 kHz; τshort3 = 0.16 ms; and (right) determined from 64 min of single-channel recording filtered at 100 Hz. The mean duration in milliseconds (and relative frequency) of Clong, Cshort1, and Cshort2 were 60,000 (0.3), 200 (0.06), and 4.7 (0.64), respectively. Open time histogram for single KCNKØ-S270LΔ872-1001 channels were determined from 18 s of single channel open burst recording, filtered at 3 kHz, τo = 2.4 s (not shown). (F) Normalized wild-type (WT) and KCNKØ-S270LΔ872-1001 (M) channel currents in groups of five to eight oocytes after 20-min incubation with 50 nM PMA or 10-min exposure to IBMX (1 mM) and forskolin (20 μM). Bars are mean ± SEM for experimental compared with initial current level.

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