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Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K+ Channels.

He K, McCord MC, Hartnett KA, Aizenman E - PLoS ONE (2015)

Bottom Line: Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue.Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant.We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine, E1456 BST, 3500 Terrace St., Pittsburgh, PA, 15261, United States of America.

ABSTRACT
Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.

No MeSH data available.


Related in: MedlinePlus

(A) Suppression of apoptotic current enhancement in Kv2.1(C73A)- but not Kv2.1(C710A)-expressing K+channels. Representative whole-cell K+ currents and pooled mean ± SEM current densities recorded from Kv2.1-expressing CHO cells without (n = 10) or with (n = 6) 30 μM DTDP, Kv2.1C73A-expressing CHO cells without (n = 9) or with (n = 7) 30 μM DTDP, or Kv2.1C710A-expressing CHO cells without (n = 9) or with (n = 12) 30 μM DTDP. Following DTDP exposure, cells were maintained in the presence of the broad spectrum protease inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF; 10 μM)-containing medium to enhance viability and facilitate electrophysiological recordings [21]. Results show that expression of the C73A channel mutant, but not the C710A mutant, prevents the increase in Kv2.1-mediated K+ currents triggered by DTDP. Currents were evoked by a series voltage steps from -80 mV to +80 mV, in 10 mV increments. To determine current density values, steady-state current amplitudes were measured 180 msec after the initiation of the +10 mV step and normalized to cell capacitance. Scale bars: 5 nA, 25 msec; **p<0.01, ***p<0.001, ANOVA/Bonferroni. (B) Kv2.1WT- and mutants-transfected CHO cells were treated with 30 μM DTDP for 10 min and incubated BAF (10 μM)-containing culture medium for 30–60 min. Total lysates were collected and ran for western blots (top). The membranes were co-probed with Kv2.1 and p-Kv2.1(S800) antibodies. GAPDH protein was used as a loading control. The levels of p-Kv2.1(S800) were calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the levels of p-Kv2.1(S800) in vehicle-treated controls, respectively (bottom). DTDP-induced phosphorylation of both cysteine mutants was inhibited, when compared to wild type Kv2.1 channels. The values represent mean ± SEM from 4 independent experiments (**p < 0.01, ANOVA/Dunnett).
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pone.0129498.g005: (A) Suppression of apoptotic current enhancement in Kv2.1(C73A)- but not Kv2.1(C710A)-expressing K+channels. Representative whole-cell K+ currents and pooled mean ± SEM current densities recorded from Kv2.1-expressing CHO cells without (n = 10) or with (n = 6) 30 μM DTDP, Kv2.1C73A-expressing CHO cells without (n = 9) or with (n = 7) 30 μM DTDP, or Kv2.1C710A-expressing CHO cells without (n = 9) or with (n = 12) 30 μM DTDP. Following DTDP exposure, cells were maintained in the presence of the broad spectrum protease inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF; 10 μM)-containing medium to enhance viability and facilitate electrophysiological recordings [21]. Results show that expression of the C73A channel mutant, but not the C710A mutant, prevents the increase in Kv2.1-mediated K+ currents triggered by DTDP. Currents were evoked by a series voltage steps from -80 mV to +80 mV, in 10 mV increments. To determine current density values, steady-state current amplitudes were measured 180 msec after the initiation of the +10 mV step and normalized to cell capacitance. Scale bars: 5 nA, 25 msec; **p<0.01, ***p<0.001, ANOVA/Bonferroni. (B) Kv2.1WT- and mutants-transfected CHO cells were treated with 30 μM DTDP for 10 min and incubated BAF (10 μM)-containing culture medium for 30–60 min. Total lysates were collected and ran for western blots (top). The membranes were co-probed with Kv2.1 and p-Kv2.1(S800) antibodies. GAPDH protein was used as a loading control. The levels of p-Kv2.1(S800) were calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the levels of p-Kv2.1(S800) in vehicle-treated controls, respectively (bottom). DTDP-induced phosphorylation of both cysteine mutants was inhibited, when compared to wild type Kv2.1 channels. The values represent mean ± SEM from 4 independent experiments (**p < 0.01, ANOVA/Dunnett).

Mentions: In previous studies [21, 22], we reported that the phosphorylation of Kv2.1 by Src and p38 kinases is associated with oxidant-triggered apoptotic K+ current surges in transfected CHO cells. Moreover, mutation of either the Y124 or S800 into non-phosphorylatable residues was sufficient to prevent Kv2.1 current enhancement following oxidant treatment. Moreover, using an alkylatable, extracellular cysteine-containing mutant channel (I379C), we were also able to demonstrate that a serine to alanine mutation at residue S800 prevented the membrane insertion of Kv2.1 following injury [22]. Since mutations at C73 or C710 could differentially prevent p38 phosphorylation of the S800 residues, we evaluated whether CHO cells expressing the C73A and C710A cysteine mutant channels would also fail to manifest an apoptotic K+ current surge. CHO cells expressing either wild type Kv2.1 or either Kv2.1(C73A) or Kv2.1(C710A) were exposed to a 10 minute treatment with the thiol oxidizing agent 2,2’-dithiodipyridine (DTDP; 30 μM), a treatment that results in a pronounced increase in K+ currents approximately 3 hours post-treatment [5, 20]. As expected, Kv2.1-expressing cells showed enhanced whole-cell K+ currents following DTDP exposure (Fig 5A). In contrast, CHO cells transfected with Kv2.1(C73A) mutant channels failed to express an increase in K+ currents, even though basal currents were comparable to those observed in WT-expressing cells (Fig 5A). This finding was consistent with the limited phosphorylation by p38 observed in this mutant (Fig 4A). Surprisingly, cells expressing Kv2.1(C710A) channels behaved similarly to WT channels. As such, we further compared the level of DTDP-induced phosphorylated S800 in CHO cells transfected with wild type Kv2.1 to S800 phosphorylation levels in cells expressing either Kv2.1(C73A) or Kv2.1(C710A). Here, we found that in both mutant Kv2.1-expressing cells, the amount S800 phosphorylation following DTDP exposure was completely blocked, when compared to wild-type expressing cells (Fig 5B). These results are in line with the results shown in Fig 4A, but cannot account for the lack of DTDP-induced current enhancement observed in the Kv2.1(C710A) mutant. It is possible that this mutation alters the conformation of the channel sufficiently to promote membrane insertion following Y124 phosphorylation but without a concomitant S800 phosphorylation. Future studies will address this possibility.


Regulation of Pro-Apoptotic Phosphorylation of Kv2.1 K+ Channels.

He K, McCord MC, Hartnett KA, Aizenman E - PLoS ONE (2015)

(A) Suppression of apoptotic current enhancement in Kv2.1(C73A)- but not Kv2.1(C710A)-expressing K+channels. Representative whole-cell K+ currents and pooled mean ± SEM current densities recorded from Kv2.1-expressing CHO cells without (n = 10) or with (n = 6) 30 μM DTDP, Kv2.1C73A-expressing CHO cells without (n = 9) or with (n = 7) 30 μM DTDP, or Kv2.1C710A-expressing CHO cells without (n = 9) or with (n = 12) 30 μM DTDP. Following DTDP exposure, cells were maintained in the presence of the broad spectrum protease inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF; 10 μM)-containing medium to enhance viability and facilitate electrophysiological recordings [21]. Results show that expression of the C73A channel mutant, but not the C710A mutant, prevents the increase in Kv2.1-mediated K+ currents triggered by DTDP. Currents were evoked by a series voltage steps from -80 mV to +80 mV, in 10 mV increments. To determine current density values, steady-state current amplitudes were measured 180 msec after the initiation of the +10 mV step and normalized to cell capacitance. Scale bars: 5 nA, 25 msec; **p<0.01, ***p<0.001, ANOVA/Bonferroni. (B) Kv2.1WT- and mutants-transfected CHO cells were treated with 30 μM DTDP for 10 min and incubated BAF (10 μM)-containing culture medium for 30–60 min. Total lysates were collected and ran for western blots (top). The membranes were co-probed with Kv2.1 and p-Kv2.1(S800) antibodies. GAPDH protein was used as a loading control. The levels of p-Kv2.1(S800) were calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the levels of p-Kv2.1(S800) in vehicle-treated controls, respectively (bottom). DTDP-induced phosphorylation of both cysteine mutants was inhibited, when compared to wild type Kv2.1 channels. The values represent mean ± SEM from 4 independent experiments (**p < 0.01, ANOVA/Dunnett).
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pone.0129498.g005: (A) Suppression of apoptotic current enhancement in Kv2.1(C73A)- but not Kv2.1(C710A)-expressing K+channels. Representative whole-cell K+ currents and pooled mean ± SEM current densities recorded from Kv2.1-expressing CHO cells without (n = 10) or with (n = 6) 30 μM DTDP, Kv2.1C73A-expressing CHO cells without (n = 9) or with (n = 7) 30 μM DTDP, or Kv2.1C710A-expressing CHO cells without (n = 9) or with (n = 12) 30 μM DTDP. Following DTDP exposure, cells were maintained in the presence of the broad spectrum protease inhibitor 1-3-Boc-aspartyl(Ome)-fluoromethyl ketone (BAF; 10 μM)-containing medium to enhance viability and facilitate electrophysiological recordings [21]. Results show that expression of the C73A channel mutant, but not the C710A mutant, prevents the increase in Kv2.1-mediated K+ currents triggered by DTDP. Currents were evoked by a series voltage steps from -80 mV to +80 mV, in 10 mV increments. To determine current density values, steady-state current amplitudes were measured 180 msec after the initiation of the +10 mV step and normalized to cell capacitance. Scale bars: 5 nA, 25 msec; **p<0.01, ***p<0.001, ANOVA/Bonferroni. (B) Kv2.1WT- and mutants-transfected CHO cells were treated with 30 μM DTDP for 10 min and incubated BAF (10 μM)-containing culture medium for 30–60 min. Total lysates were collected and ran for western blots (top). The membranes were co-probed with Kv2.1 and p-Kv2.1(S800) antibodies. GAPDH protein was used as a loading control. The levels of p-Kv2.1(S800) were calculated from the ratio of p-Kv2.1(S800) to total Kv2.1 protein, and then normalized to the levels of p-Kv2.1(S800) in vehicle-treated controls, respectively (bottom). DTDP-induced phosphorylation of both cysteine mutants was inhibited, when compared to wild type Kv2.1 channels. The values represent mean ± SEM from 4 independent experiments (**p < 0.01, ANOVA/Dunnett).
Mentions: In previous studies [21, 22], we reported that the phosphorylation of Kv2.1 by Src and p38 kinases is associated with oxidant-triggered apoptotic K+ current surges in transfected CHO cells. Moreover, mutation of either the Y124 or S800 into non-phosphorylatable residues was sufficient to prevent Kv2.1 current enhancement following oxidant treatment. Moreover, using an alkylatable, extracellular cysteine-containing mutant channel (I379C), we were also able to demonstrate that a serine to alanine mutation at residue S800 prevented the membrane insertion of Kv2.1 following injury [22]. Since mutations at C73 or C710 could differentially prevent p38 phosphorylation of the S800 residues, we evaluated whether CHO cells expressing the C73A and C710A cysteine mutant channels would also fail to manifest an apoptotic K+ current surge. CHO cells expressing either wild type Kv2.1 or either Kv2.1(C73A) or Kv2.1(C710A) were exposed to a 10 minute treatment with the thiol oxidizing agent 2,2’-dithiodipyridine (DTDP; 30 μM), a treatment that results in a pronounced increase in K+ currents approximately 3 hours post-treatment [5, 20]. As expected, Kv2.1-expressing cells showed enhanced whole-cell K+ currents following DTDP exposure (Fig 5A). In contrast, CHO cells transfected with Kv2.1(C73A) mutant channels failed to express an increase in K+ currents, even though basal currents were comparable to those observed in WT-expressing cells (Fig 5A). This finding was consistent with the limited phosphorylation by p38 observed in this mutant (Fig 4A). Surprisingly, cells expressing Kv2.1(C710A) channels behaved similarly to WT channels. As such, we further compared the level of DTDP-induced phosphorylated S800 in CHO cells transfected with wild type Kv2.1 to S800 phosphorylation levels in cells expressing either Kv2.1(C73A) or Kv2.1(C710A). Here, we found that in both mutant Kv2.1-expressing cells, the amount S800 phosphorylation following DTDP exposure was completely blocked, when compared to wild-type expressing cells (Fig 5B). These results are in line with the results shown in Fig 4A, but cannot account for the lack of DTDP-induced current enhancement observed in the Kv2.1(C710A) mutant. It is possible that this mutation alters the conformation of the channel sufficiently to promote membrane insertion following Y124 phosphorylation but without a concomitant S800 phosphorylation. Future studies will address this possibility.

Bottom Line: Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue.Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant.We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine, E1456 BST, 3500 Terrace St., Pittsburgh, PA, 15261, United States of America.

ABSTRACT
Caspase activity during apoptosis is inhibited by physiological concentrations of intracellular K+. To enable apoptosis in injured cortical and hippocampal neurons, cellular loss of this cation is facilitated by the insertion of Kv2.1 K+ channels into the plasma membrane via a Zn2+/CaMKII/SNARE-dependent process. Pro-apoptotic membrane insertion of Kv2.1 requires the dual phosphorylation of the channel by Src and p38 at cytoplasmic N- and C-terminal residues Y124 and S800, respectively. In this study, we investigate if these phosphorylation sites are mutually co-regulated, and whether putative N- and C-terminal interactions, possibly enabled by Kv2.1 intracellular cysteine residues C73 and C710, influence the phosphorylation process itself. Studies were performed with recombinant wild type and mutant Kv2.1 expressed in Chinese hamster ovary (CHO) cells. Using immunoprecipitated Kv2.1 protein and phospho-specific antibodies, we found that an intact Y124 is required for p38 phosphorylation of S800, and, importantly, that Src phosphorylation of Y124 facilitates the action of the p38 at the S800 residue. Moreover, the actions of Src on Kv2.1 are substantially decreased in the non-phosphorylatable S800A channel mutant. We also observed that mutations of either C73 or C710 residues decreased the p38 phosphorylation at S800 without influencing the actions of Src on tyrosine phosphorylation of Kv2.1. Surprisingly, however, apoptotic K+ currents were suppressed only in cells expressing the Kv2.1(C73A) mutant but not in those transfected with Kv2.1(C710A), suggesting a possible structural alteration in the C-terminal mutant that facilitates membrane insertion. These results show that intracellular N-terminal domains critically regulate phosphorylation of the C-terminal of Kv2.1, and vice versa, suggesting possible new avenues for modifying the apoptotic insertion of these channels during neurodegenerative processes.

No MeSH data available.


Related in: MedlinePlus