Manville RW and Abbott GW (2021), The Amyloid Precursor Protein C99 Fragment Modu...

XB-ART-58331

Cell Physiol Biochem 2021 Jul 28;55S3:157-170. doi: 10.33594/000000397.

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The Amyloid Precursor Protein C99 Fragment Modulates Voltage-Gated Potassium Channels.

Manville RW , Abbott GW .

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BACKGROUND/AIMS: The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel β subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels. METHODS: We quantified the effects of C99 on Kv channel function, using electrophysiological analysis of subunits expressed in Xenopus laevis oocytes, biochemical and immunofluorescence techniques. RESULTS: C99 isoform-selectively inhibited (by 30-80%) activity of a range of Kv channels. Among the KCNQ (Kv7) family, C99 isoform-selectively inhibited, shifted the voltage dependence and/or slowed activation of KCNQ2, KCNQ3, KCNQ2/3 and KCNQ5, with no effects on KCNQ1, KCNQ1-KCNE1 or KCNQ4. C99/APP co-localized with KCNQ2 and KCNQ3 in adult rat sciatic nerve nodes of Ranvier. Both C99 and full-length APP co-immunoprecipitated with KCNQ2 in vitro, yet unlike C99, APP only weakly affected KCNQ2/3 activity. Finally, C99 altered the effects on KCNQ2/3 function of inhibitors tetraethylammounium and XE991, but not openers retigabine and ICA27243. CONCLUSION: Our findings raise the possibility of C99 accumulation early in AD altering cellular excitability by modulating Kv channel activity.

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Species referenced: Xenopus laevis
Genes referenced: app kcne1 kcne2 kcnq1 kcnq2 kcnq3 kcnq4 kcnq5

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	Fig. 2. C99 isoform-selectively regulates KCNQ channel function. All error bars indicate SEM. Dashed lines indicate zero current level. A: Left, current/voltage (I/V) relationships; right, normalized tail current versus prepulse voltage (G/Gmax), for KCNQ1 alone (black; n = 11) or with C99 (red; n = 20) quantified by TEVC. Right inset, voltage protocol used for all similar data in this study. B: Data as in A for KCNQ1-KCNE1 (Q1-E1) alone or with C99; n = 9–10. C: Data as in A for KCNQ4 alone or with C99; n = 8–15. D: Averaged current traces for KCNQ2 alone or with C99; n = 20. E: Left, mean peak current versus voltage; right, G/Gmax, for KCNQ2 alone (black) or with C99 (blue); n = 20. *P = 0.002 for KCNQ2 versus KCNQ2-C99. F: Averaged current traces for KCNQ3 alone or with C99; n = 9–12. G: Left, mean peak current versus voltage; center, raw tail current versus prepulse voltage; right, G/Gmax, for KCNQ3* alone (black) or with C99 (blue); n = 9–12. H: Averaged current traces for KCNQ5 alone or with C99; n = 10. I: Left, mean peak current versus voltage; center, raw tail current versus prepulse voltage; right, G/Gmax, for KCNQ5 alone (black) or with C99 (blue); n = 10. J: Scatter plot showing effects of KCNQ3* alone or with C99 on unclamped oocyte membrane potential (EM); n = 9–12. K: Mean effects of C99 on KCNQ3* activation rate; n = 9–12. #P <0.0001. L: Lack of effects of C99 on KCNQ3* mean deactivation rate at −80 mV following different duration pulses to +40 mV; n = 10–12. #P >0.05. M: Scatter plot showing effects of KCNQ3* alone or with C99 on unclamped oocyte membrane potential (EM); n = 10.
	Fig. 3. C99 has varied effects on other Kv channels. All error bars indicate SEM, all statistics at +40 mV. Mean peak current versus voltage relationships (left) and G/Gmax (right; except panel C) from traces recorded by TEVC in Xenopus oocytes expressing Kv channels alone (black) or with C99 (red); Voltage protocol, upper center inset. A: Kv1.1 with/without C99; n = 7–8; **P = 3.4×10−5. B: Kv1.2 with/without C99; n = 20–25; P = 0.01. C: Kv4 channels as indicated with/without C99; n = 8–21; P<0.05. D: hERG with/without C99; n = 22–23; **P = 0.002.
	Fig. 4. C99 regulates KCNQ2/3 channel function. All error bars indicate SEM. Dashed lines indicate zero current level. A: Mean current traces for KCNQ2/3 alone (black) or with C99 (red) quantified by TEVC using voltage protocol inset. B: Mean current/voltage (I/V) relationships for KCNQ2/3 alone (black; n = 25) or with C99 (red; n = 30) quantified by TEVC. **P = 0.00003 at +40 mV. C: Mean normalized tail current versus prepulse voltage (G/Gmax), for KCNQ2/3 alone (black; n = 25) or with C99 (red; n = 30) quantified by TEVC. D: Lack of effects of C99 on KCNQ2/3 ion selectivity quantified by measuring various ion permeabilities relative to that of K+; n = 10–12. E: Mean activation rate for KCNQ2/3 alone or with C99 as indicated; n = 25–30. P = 0.006.
	Fig. 5. C99/APP co-localizes with KCNQ2/3 in rat sciatic nerves and co-assembles with KCNQ2/3 in vitro. A: Representative (from at least two different days, and >2 sections per day, for each combination) fluorescence images of adult rat frozen sciatic nerve nodes of Ranvier double-immunostained using antibodies and colors as indicated. Merge indicates merged image of two color channels. Scale bars = 2 μm. Left, KCNQ2 and C99; center, KCNQ2 and Ankyrin G (nodal marker); right, KCNQ3 and APP. B: Western blot showing co-immunopreciptation of N-terminal flag-tagged C99 (C99NT-flag) with KCNQ2 (Q2), subunit expression indicated above with + symbols. IP, immunoprecipitation; IB, immunoblot. C: Western blot showing co-immunopreciptation of KCNQ2 (Q2) with N- and C-terminal flag-tagged C99 and with APP, but not untagged C99 (when co-IPing with anti-flag antibody). Subunit expression indicated above with + symbols. IP, immunoprecipitation; IB, immunoblot. D: Western blot showing co-immunopreciptation of KCNQ2 (Q2) with APP (using anti-APP antibody), but not untagged C99 (when co-IPing with anti-flag antibody); as a positive control KCNQ2 was IPed with anti-KCNQ2 antibody. Subunit expression indicated above with + symbols. IP, immunoprecipitation; IB, immunoblot; lys, lysate control. E: Left, mean peak current/voltage (I/V); right, mean G/Gmax relationships, for KCNQ2/3 alone (black; n = 12) or with synthetic cDNA construct derived subunits as follows: wild-type C99 (red; n = 16), C-terminal flag-tagged C99 (red; n = 13), N-terminal-tagged C99 (red; n = 11), wild-type APP (red; n = 17) quantified by TEVC. P = 0.05; *P = 0.01 versus KCNQ2/3 alone at +40 mV; others P>0.05. Voltage protocol, upper left inset.
	Fig. 6. C99 does not prevent KCNQ2/3 current potentiation by pharmacological openers. All error bars indicate SEM. Dashed lines indicate zero current level. A: Upper, retigabine structure; lower, in silico docking of retigabine close to KCNQ3-W265. B: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 10 μM retigabine (RTG) (n = 6–10). Voltage protocol, center inset. C: Mean tail current (left) and normalized tail currents (G/Gmax; right) versus prepulse voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 10 μM RTG as indicated (n = 6–10). D: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 10 μM ICA27243 (n = 5–8). Voltage protocol as in B. E: Mean peak current (left) and normalized tail currents (G/Gmax; right) versus prepulse voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 10 μM ICA27243 as indicated (n = 5–8). F: Mean effects of 10 μM ICA27243 on the activation rate of KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 10 μM ICA27243 as indicated (n = 5–8).
	Fig. 7. C99 alters the effects of inhibitors on KCNQ2/3. All error bars indicate SEM. Dashed lines indicate zero current level. A: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 98 mM TEA (n = 6–8). Voltage protocol, center inset. B: Mean peak current versus voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 98 mM TEA (n = 6–8) for traces as in A. C: Mean peak current inhibition by TEA of KCNQ2/3 alone (green) or with C99 (purple) for traces as in A; n = 6–8; # P <0.0001. D: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 50 μM XE991 (n = 8–10). Voltage protocol, center inset. E: Mean peak current versus voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 50 μM XE991 (n = 8–10) for traces as in D. F: Mean peak current inhibition versus voltage by 50 μM XE991 of KCNQ2/3 alone (green) or with C99 (purple) for traces as in D; n = 8–10; # P <0.0001.

References [+] :

Abbott, KCNQ1, KCNE2, and Na+-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability. 2014, Pubmed, Xenbase

	Fig. 2. C99 isoform-selectively regulates KCNQ channel function. All error bars indicate SEM. Dashed lines indicate zero current level. A: Left, current/voltage (I/V) relationships; right, normalized tail current versus prepulse voltage (G/Gmax), for KCNQ1 alone (black; n = 11) or with C99 (red; n = 20) quantified by TEVC. Right inset, voltage protocol used for all similar data in this study. B: Data as in A for KCNQ1-KCNE1 (Q1-E1) alone or with C99; n = 9–10. C: Data as in A for KCNQ4 alone or with C99; n = 8–15. D: Averaged current traces for KCNQ2 alone or with C99; n = 20. E: Left, mean peak current versus voltage; right, G/Gmax, for KCNQ2 alone (black) or with C99 (blue); n = 20. *P = 0.002 for KCNQ2 versus KCNQ2-C99. F: Averaged current traces for KCNQ3 alone or with C99; n = 9–12. G: Left, mean peak current versus voltage; center, raw tail current versus prepulse voltage; right, G/Gmax, for KCNQ3* alone (black) or with C99 (blue); n = 9–12. H: Averaged current traces for KCNQ5 alone or with C99; n = 10. I: Left, mean peak current versus voltage; center, raw tail current versus prepulse voltage; right, G/Gmax, for KCNQ5 alone (black) or with C99 (blue); n = 10. J: Scatter plot showing effects of KCNQ3* alone or with C99 on unclamped oocyte membrane potential (EM); n = 9–12. K: Mean effects of C99 on KCNQ3* activation rate; n = 9–12. #P <0.0001. L: Lack of effects of C99 on KCNQ3* mean deactivation rate at −80 mV following different duration pulses to +40 mV; n = 10–12. #P >0.05. M: Scatter plot showing effects of KCNQ3* alone or with C99 on unclamped oocyte membrane potential (EM); n = 10.
	Fig. 3. C99 has varied effects on other Kv channels. All error bars indicate SEM, all statistics at +40 mV. Mean peak current versus voltage relationships (left) and G/Gmax (right; except panel C) from traces recorded by TEVC in Xenopus oocytes expressing Kv channels alone (black) or with C99 (red); Voltage protocol, upper center inset. A: Kv1.1 with/without C99; n = 7–8; **P = 3.4×10−5. B: Kv1.2 with/without C99; n = 20–25; P = 0.01. C: Kv4 channels as indicated with/without C99; n = 8–21; P<0.05. D: hERG with/without C99; n = 22–23; **P = 0.002.
	Fig. 4. C99 regulates KCNQ2/3 channel function. All error bars indicate SEM. Dashed lines indicate zero current level. A: Mean current traces for KCNQ2/3 alone (black) or with C99 (red) quantified by TEVC using voltage protocol inset. B: Mean current/voltage (I/V) relationships for KCNQ2/3 alone (black; n = 25) or with C99 (red; n = 30) quantified by TEVC. **P = 0.00003 at +40 mV. C: Mean normalized tail current versus prepulse voltage (G/Gmax), for KCNQ2/3 alone (black; n = 25) or with C99 (red; n = 30) quantified by TEVC. D: Lack of effects of C99 on KCNQ2/3 ion selectivity quantified by measuring various ion permeabilities relative to that of K+; n = 10–12. E: Mean activation rate for KCNQ2/3 alone or with C99 as indicated; n = 25–30. P = 0.006.
	Fig. 5. C99/APP co-localizes with KCNQ2/3 in rat sciatic nerves and co-assembles with KCNQ2/3 in vitro. A: Representative (from at least two different days, and >2 sections per day, for each combination) fluorescence images of adult rat frozen sciatic nerve nodes of Ranvier double-immunostained using antibodies and colors as indicated. Merge indicates merged image of two color channels. Scale bars = 2 μm. Left, KCNQ2 and C99; center, KCNQ2 and Ankyrin G (nodal marker); right, KCNQ3 and APP. B: Western blot showing co-immunopreciptation of N-terminal flag-tagged C99 (C99NT-flag) with KCNQ2 (Q2), subunit expression indicated above with + symbols. IP, immunoprecipitation; IB, immunoblot. C: Western blot showing co-immunopreciptation of KCNQ2 (Q2) with N- and C-terminal flag-tagged C99 and with APP, but not untagged C99 (when co-IPing with anti-flag antibody). Subunit expression indicated above with + symbols. IP, immunoprecipitation; IB, immunoblot. D: Western blot showing co-immunopreciptation of KCNQ2 (Q2) with APP (using anti-APP antibody), but not untagged C99 (when co-IPing with anti-flag antibody); as a positive control KCNQ2 was IPed with anti-KCNQ2 antibody. Subunit expression indicated above with + symbols. IP, immunoprecipitation; IB, immunoblot; lys, lysate control. E: Left, mean peak current/voltage (I/V); right, mean G/Gmax relationships, for KCNQ2/3 alone (black; n = 12) or with synthetic cDNA construct derived subunits as follows: wild-type C99 (red; n = 16), C-terminal flag-tagged C99 (red; n = 13), N-terminal-tagged C99 (red; n = 11), wild-type APP (red; n = 17) quantified by TEVC. P = 0.05; *P = 0.01 versus KCNQ2/3 alone at +40 mV; others P>0.05. Voltage protocol, upper left inset.
	Fig. 6. C99 does not prevent KCNQ2/3 current potentiation by pharmacological openers. All error bars indicate SEM. Dashed lines indicate zero current level. A: Upper, retigabine structure; lower, in silico docking of retigabine close to KCNQ3-W265. B: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 10 μM retigabine (RTG) (n = 6–10). Voltage protocol, center inset. C: Mean tail current (left) and normalized tail currents (G/Gmax; right) versus prepulse voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 10 μM RTG as indicated (n = 6–10). D: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 10 μM ICA27243 (n = 5–8). Voltage protocol as in B. E: Mean peak current (left) and normalized tail currents (G/Gmax; right) versus prepulse voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 10 μM ICA27243 as indicated (n = 5–8). F: Mean effects of 10 μM ICA27243 on the activation rate of KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 10 μM ICA27243 as indicated (n = 5–8).
	Fig. 7. C99 alters the effects of inhibitors on KCNQ2/3. All error bars indicate SEM. Dashed lines indicate zero current level. A: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 98 mM TEA (n = 6–8). Voltage protocol, center inset. B: Mean peak current versus voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 98 mM TEA (n = 6–8) for traces as in A. C: Mean peak current inhibition by TEA of KCNQ2/3 alone (green) or with C99 (purple) for traces as in A; n = 6–8; # P <0.0001. D: Mean TEVC traces for KCNQ2/3 with or without C99 in the absence (Control) or presence of 50 μM XE991 (n = 8–10). Voltage protocol, center inset. E: Mean peak current versus voltage relationships recorded by TEVC in Xenopus oocytes expressing KCNQ2/3 alone or with C99 in the absence (black) or presence (red) of 50 μM XE991 (n = 8–10) for traces as in D. F: Mean peak current inhibition versus voltage by 50 μM XE991 of KCNQ2/3 alone (green) or with C99 (purple) for traces as in D; n = 8–10; # P <0.0001.