Tong Y , Brandt GS , Li M , Shapovalov G , Slimko E , Karschin A , Dougherty DA , Lester HA .

GM29836 NIGMS NIH HHS , NS34407 NINDS NIH HHS , R01 NS034407 NINDS NIH HHS , R37 NS034407 NINDS NIH HHS , Wellcome Trust

	Figure 1. Topology of Kir2.1 from Mus musculus (GenBank accession number X73052). The sequence surrounding the tyrosine-242 contains consensus sequences for both tyrosine phosphorylation and tyrosine-based interaction with clathrin adaptor proteins. Other consensus endocytosis sequences are also shown.
	Figure 2. Whole-cell current from oocytes expressing Kir2.1 (A) and Kir2.1-Y242F (B). Data columns represent normalized currents (mean ± SEM, 4–5 oocytes). Where indicated, oocytes were coinjected with cRNA for tyrosine kinases v-Src or PyK2 and incubated with 10 μM PAO for at least 30 min before recording. 100% corresponds to 13.3 μA in A and 31.4 μA in B.
	Figure 3. Conditions favoring tyrosine phosphorylation do not change single-channel conductance of WT Kir2.1. (A) Traces showing exemplar single-channel currents from cell-attached patches at −100 mV. The arrows point to the closed states and to the open states for control oocytes (left) and for oocytes coinjected with v-Src and treated with PAO (right). (B) Normalized all-points amplitude histograms for exemplar patches. Data from an oocyte injected with Kir2.1 alone are shown as heavy lines, and data from an oocyte injected with Kir2.1 + v-Src and exposed to PAO are shown as light lines.
	Figure 4. Inhibition of current and decrease of capacitance in oocytes expressing Kir2.1-Y242TAG suppressed with Tyr(ONB). (A) Typical current traces from individual oocytes. The left panel shows the effect of irradiating an oocyte (3 s at arrow) that is neither coexpressing v-Src nor exposed to PAO. The center panel shows the effect of coexpression of v-Src and exposure to PAO, but no irradiation of the oocyte. The right panel shows traces from an oocyte coexpressing v-Src, exposed to PAO, and irradiated. Oocytes were clamped at 0 mV and stepped to −80 mV to elicit an inward current. Bath solutions contained 96 mM KCl. Time after irradiation is shown. (B) Plots of current and capacitance versus time from the individual oocytes in A. (C) Averaged data from batches of oocytes recorded at t = 30 min. The filled bars correspond to the left-hand y-axis, indicating normalized current decrease. Average values (and ranges) for 100% current were 8.7 (5.5–13), 6.5 (4.5–10.4), and 6.6 (1.6–15.5) μA for the three groups (left to right). The hollow bars correspond to the right-hand y-axis, indicating capacitance decrease. Average values (and ranges) for 100% capacitance were 190 (159–227), 187 (167–202), and 194 (132–234) nF for the three groups (left to right). Error bars indicate SEM, n = 4–6 oocytes.
	Figure 5. Dynamin expression distinguishes inhibition of the channel from endocytosis. (A) Current and capacitance data from representative oocytes coexpressing v-Src and Kir2.1-Y242TAG suppressed with Tyr(ONB) and treated with PAO. Data were recorded 48 h after injection. (Left) An oocyte without coinjected dynamin cRNA. (Center) An oocyte coinjected with 10 ng WT dynamin cRNA. The capacitance decrease for this cell was unusually large. (Right) An oocyte coinjected with 10 ng dominant negative dynamin-K44A. (B) Data for current and capacitance for oocytes recorded 30 min after irradiation (mean ± SEM, n = 5). Left-hand y-axis and full bars represent normalized current decrease. Average values (and ranges) for 100% were 4.1 (3.15–4.8), 3.1 (2.8–3.7), and 4.1 (3.0–5.7) μA for the three groups (left to right). Right-hand axis corresponds to hollow bars, representing normalized capacitance decrease. The rightmost column shows capacitance change of uninjected control oocytes. Average values (and ranges) for 100% capacitance were 197 (191–205), 225 (191–248), 189 (174–199), and 197 (190–209) nF for the four groups (left to right). Error bars represent SEM.
	Figure 6. Fluorescence analysis of membrane retrieval. (A) Confocal image of two oocytes labeled with 10 μM tetramethylrhodamine-5-maleimide, a membrane-impermeant thiol-reactive fluorophore. The oocyte at left was injected with v-Src and Kir2.1-Y242TAG suppressed with Tyr(ONB). The oocyte on the right was not injected. (B) Fluorescence measurements for individual oocytes coexpressing v-Src and Kir2.1 Y242Tyr(ONB) and exposed to PAO. Oocytes were labeled with 10 μM tetramethylrhodamine-5-maleimide for 30 min before recording. Fluorescence is measured as PMT output voltage. As indicated, controls included omission of coinjected v-Src and omission of irradiation. (C) Normalized, average fluorescence change at t = 30 min for oocytes expressing Kir2.1-Y242TAG suppressed with Tyr(ONB) and treated with PAO, and then labeled with tetramethylrhodamine maleimide. The first column represents a control in which oocytes were not coinjected with v-Src. In all other columns, oocytes expressed the Kir2.1-Y242Tyr(ONB) along with v-Src. The second column represents a control in which oocytes were not irradiated. In the third column, no exogenous dynamin was added. The fourth column shows oocytes coexpressing WT dynamin. The fifth column represents oocytes coexpressing dynamin-K44A. Each bar represents six or seven oocytes. The average values (and ranges) for 100% fluorescence were 5.76 (3–9.5), 5.74 (3.5–7.2), 6.45 (3.7–9.5), 5.60 (3.93–6.92), and 5.62 (1–9.7) V.
	Figure 7. Western blotting shows that Kir2.1 appears not to be phosphorylated on tyrosine, where detection of TrkB phosphorylation serves as a control for the method. The experiment is typical of at least six similar results. (A) Western blot of membranes from oocytes expressing TrkB shows positive staining with both anti-TrkB and anti-PY antibodies. (Lane 1) Uninjected oocytes; and (lane 2) TrkB. Nitrocellulose blot is stained on the left with C14 anti-TrkB antibody, and on the right with 4G10 antiphosphotyrosine antibody. Lane 1 has been loaded with 50% more membrane protein than lane 2 (which received protein from membrane dissected from 21 oocytes), to emphasize the absence of an endogenous phosphorylated band at the position of TrkB. Arrowheads indicate TrkB. (B) Western blot of membranes from oocytes expressing epitope-tagged Kir2.1 shows positive staining with anti-HA antibody, but not with the antiphosphotyrosine antibody 4G10. (Lane 1) Uninjected oocytes; (lane 2) Kir2.1-HA; (lane 3) Kir2.1Y242F-HA; and (lane 4) Kir2.1-HA prepared by in vitro translation from wheat germ extract. Nitrocellulose blot is stained on the left with BAbCo HA.11 antihemagglutinin antibody, and on the right with 4G10 antiphosphotyrosine antibody. Arrows indicate Kir2.1. Lanes 1–3 contain protein from membranes dissected from 21–23 oocytes.
	Figure 8. Schematic interpretation of the experiments. Irradiation leads to decaging of Tyr242 of Kir 2.1. Pathway A shows direct phosphorylation of the newly revealed tyrosine, which may be reversible/transient. Failure to observe phosphotyrosine directly makes this pathway unlikely. Pathways B and C involve the μ subunit of AP-1, AP-2, or AP-3 or a similar molecule. In pathway B the adaptor protein (AP) is phosphorylated, which enables it to bind to the now available tyrosine-based interaction motif of Kir 2.1 and inhibit the channel. Endocytosis follows. Pathway C includes phosphorylation of an unspecified protein, which, in combination with the adaptor protein, binds to and inhibits Kir 2.1 and leads to endocytosis.

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