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Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

Cavaretta JP, Sherer KR, Lee KY, Kim EH, Issema RS, Chung HJ - PLoS ONE (2014)

Bottom Line: Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon.Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface.These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

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CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface.(A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca2+-insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p<0.05, **p<0.01, ***p<0.001). (F) Model by which apoCaM interaction with helices A and B of KCNQ2 and KCNQ3 is critical for trafficking of KCNQ2/KCNQ3 channels from the ER to the axonal surface.
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pone-0103655-g013: CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface.(A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca2+-insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p<0.05, **p<0.01, ***p<0.001). (F) Model by which apoCaM interaction with helices A and B of KCNQ2 and KCNQ3 is critical for trafficking of KCNQ2/KCNQ3 channels from the ER to the axonal surface.

Mentions: Coexpression of Ca2+-insensitive CaM has been reported to moderately increase the ER retention of KCNQ2 by 20% in HEK293T cells [24], [26], suggesting that enrichment of KCNQ channels at the axonal surface may be regulated by Ca2+-bound CaM. To test this, we performed surface immunostaining of HA-KCNQ3/KCNQ2 channels in cultured hippocampal neurons that were cotransfected with either wild-type CaM or dominant-negative mutant CaM1234 (Fig. 13). Although CaM1234 is unable to bind Ca2+[27], it has been shown to associate with KCNQ2 [22]. Given that CaM is abundantly expressed in cultured hippocampal neurons at 5 DIV, which is when transfections were performed (Fig. 1B), we hypothesized that coexpression of CaM1234 would displace endogenous CaM and make the CaM complex Ca2+-insensitive [27].


Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

Cavaretta JP, Sherer KR, Lee KY, Kim EH, Issema RS, Chung HJ - PLoS ONE (2014)

CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface.(A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca2+-insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p<0.05, **p<0.01, ***p<0.001). (F) Model by which apoCaM interaction with helices A and B of KCNQ2 and KCNQ3 is critical for trafficking of KCNQ2/KCNQ3 channels from the ER to the axonal surface.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4117524&req=5

pone-0103655-g013: CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface.(A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca2+-insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p<0.05, **p<0.01, ***p<0.001). (F) Model by which apoCaM interaction with helices A and B of KCNQ2 and KCNQ3 is critical for trafficking of KCNQ2/KCNQ3 channels from the ER to the axonal surface.
Mentions: Coexpression of Ca2+-insensitive CaM has been reported to moderately increase the ER retention of KCNQ2 by 20% in HEK293T cells [24], [26], suggesting that enrichment of KCNQ channels at the axonal surface may be regulated by Ca2+-bound CaM. To test this, we performed surface immunostaining of HA-KCNQ3/KCNQ2 channels in cultured hippocampal neurons that were cotransfected with either wild-type CaM or dominant-negative mutant CaM1234 (Fig. 13). Although CaM1234 is unable to bind Ca2+[27], it has been shown to associate with KCNQ2 [22]. Given that CaM is abundantly expressed in cultured hippocampal neurons at 5 DIV, which is when transfections were performed (Fig. 1B), we hypothesized that coexpression of CaM1234 would displace endogenous CaM and make the CaM complex Ca2+-insensitive [27].

Bottom Line: Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon.Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface.These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

Show MeSH
Related in: MedlinePlus