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The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

Shruti S, Urban-Ciecko J, Fitzpatrick JA, Brenner R, Bruchez MP, Barth AL - PLoS ONE (2012)

Bottom Line: Here we investigate β4's effect on BK channel trafficking to the plasma membrane.Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence.Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

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Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.
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pone-0033429-g001: Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.

Mentions: Because transient transfections with multiple plasmids does not guarantee expression of all plasmids (for example, some cells may not have taken up the β4 plasmid), analysis was limited to cells that exhibited both GFP fluorescence as well as MG-2P surface signal. This method may have led to an overestimation of surface localization of FAP-tagged BKα in the BKα+β4 transfected cells, since some cells may not have expressed the β4 construct. Adjacent membranes from two transfected cells were rejected from analysis. ROIs for 2–4 cells were analyzed per image field, and 20 fields per transfection experiment were evaluated. Thus, ∼60 cells per condition were examined for each experiment. Experiments were repeated at least three times. Mean, minimum and maximum pixel intensities for each ROI were calculated, averaged for each cell, and then averaged across cells for each separate transfection. To account for potential differences in expression levels across different transfection experiments, all transfection datasets were normalized to the value of the mean surface fluorescence for the BKα-alone transfection for that specific experimental day. A histogram for the normalized fluorescence intensity values across all experimental days was generated (see Figure 1).


The brain-specific Beta4 subunit downregulates BK channel cell surface expression.

Shruti S, Urban-Ciecko J, Fitzpatrick JA, Brenner R, Bruchez MP, Barth AL - PLoS ONE (2012)

Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3306404&req=5

pone-0033429-g001: Co-expression of the β4 subunit reduces cell-surface trafficking of BK channels.(A) Membrane topology of the FAP-BKα and β4 proteins. The FAP tag is at the extracellular, N-terminus. The C-terminus of the BKα subunit is indicated. (B) Schematic of binding of cell-impermeable dye (pink) to the FAP results in significant increase in fluorescence (red). (C) Application of cell-impermeable dye labels only surface BKα channels in live HEK-293 cells co-transfected with FAP-BKα (red) and GFP (green). (D) Same as (C) but in cells co-transfected with β4, FAP-BKα, and GFP showing reduced surface expressed of the FAP-BKα. (E) Application of cell-permeable dye labels intracellular stores of channel in cells transfected with FAP-BKα (red) and GFP (green). (F) Same as (E) but in cells transfected with β4, FAP-BKα, and GFP. Scale bar = 20 µm (C–F). (G) Distribution of surface fluorescence intensity values after application of cell-impermeable dye in transfected cells for FAP-BKα (red bars) or FAP-BKα+β4 (black bars). (H–I) Proposed model for surface distribution of BK channels in the absence (H) or presence (I) of the β4 subunit.
Mentions: Because transient transfections with multiple plasmids does not guarantee expression of all plasmids (for example, some cells may not have taken up the β4 plasmid), analysis was limited to cells that exhibited both GFP fluorescence as well as MG-2P surface signal. This method may have led to an overestimation of surface localization of FAP-tagged BKα in the BKα+β4 transfected cells, since some cells may not have expressed the β4 construct. Adjacent membranes from two transfected cells were rejected from analysis. ROIs for 2–4 cells were analyzed per image field, and 20 fields per transfection experiment were evaluated. Thus, ∼60 cells per condition were examined for each experiment. Experiments were repeated at least three times. Mean, minimum and maximum pixel intensities for each ROI were calculated, averaged for each cell, and then averaged across cells for each separate transfection. To account for potential differences in expression levels across different transfection experiments, all transfection datasets were normalized to the value of the mean surface fluorescence for the BKα-alone transfection for that specific experimental day. A histogram for the normalized fluorescence intensity values across all experimental days was generated (see Figure 1).

Bottom Line: Here we investigate β4's effect on BK channel trafficking to the plasma membrane.Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence.Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT
The large-conductance K(+) channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca(++)- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

Show MeSH
Related in: MedlinePlus