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Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S).

Sitdikova GF, Fuchs R, Kainz V, Weiger TM, Hermann A - Front Physiol (2014)

Bottom Line: The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution.A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology of Man and Animals, Kazan Federal University Kazan, Russia.

ABSTRACT

Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity.

Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.

Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode.

Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.

Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

No MeSH data available.


Related in: MedlinePlus

Mean values of BK channel open time probabilities (Popen). Three types of channels, GH3-BK (A), GH4-BK (B), and GH4-STREX (C) cells, were investigated. Bar graphs show Po-values under control conditions (left bars, white) and after extracellular application of NaHS (right bars, gray). ATP, kinase, kinase inhibitor, kinase inhibitor staurosporine (STS), or phosphatase blocker okadaic acid (OA) were added to the pipette solution; ATP0 – no ATP added; ATP1 – 1 mM ATP added; PKA (50 units); PKCsu (0.1 units/ml); PKCin – 500 nM; STS – 1 μM; OA – 100 nM. Figures are based on back transformed means and s.e.m. values (error bars), respectively. Asteriks indicate significance of H2S (300 μM NaHS equivalent to effective 34–41 μM H2S) effects according to the logit transformed data.
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Figure 3: Mean values of BK channel open time probabilities (Popen). Three types of channels, GH3-BK (A), GH4-BK (B), and GH4-STREX (C) cells, were investigated. Bar graphs show Po-values under control conditions (left bars, white) and after extracellular application of NaHS (right bars, gray). ATP, kinase, kinase inhibitor, kinase inhibitor staurosporine (STS), or phosphatase blocker okadaic acid (OA) were added to the pipette solution; ATP0 – no ATP added; ATP1 – 1 mM ATP added; PKA (50 units); PKCsu (0.1 units/ml); PKCin – 500 nM; STS – 1 μM; OA – 100 nM. Figures are based on back transformed means and s.e.m. values (error bars), respectively. Asteriks indicate significance of H2S (300 μM NaHS equivalent to effective 34–41 μM H2S) effects according to the logit transformed data.

Mentions: Experimental values of BK single channel open probability (Po) for GH3, GH4, and GH4 STREX cells are listed in Table 2. Po-values are presented as mean ± standard error of mean. ATP was added to the pipette solution (intracellular membrane face). In a physiological pipette solution containing no ATP (assigned ATP0), BK single channel open probability (Po) was generally low. Addition of 1 mM ATP (ATP1) to the pipette solution most significantly increased Po (***) (Table 2 and Figure 3A). Similar findings have been reported previously from GH3 cells (Denson et al., 2001; Zhou et al., 2012), and reviewed for other cell types (Schubert and Nelson, 2001). All further solutions contained 1 mM ATP. PKA catalytic subunit (50 units/ml) altered Po, but both values were non-significant compared to ATP1 (Figure 3A), however, they were both significantly (***) different to ATP0. PKC (PKC catalytic subunit, PKCsu, 0.1 units/ml) in the pipette solution decreased BK Po non-significantly compared to ATP1, but Po was significantly different from ATP0 (*). PKC is known to cause inhibition of BK channel activity from GH cells (Shipston and Armstrong, 1996; Hall and Armstrong, 2000; Wu et al., 2007; Zhou et al., 2012, 2010). With protein kinase inhibitor pseudo-substrate (PKCin, fragment 19–31, 500 nM) Po increased non-significantly if compared to PKCsu or ATP1, but was significantly higher than during ATP0 conditions (**) if compared to ATP1. The experiments support previous results of an inhibitory role of PKC at BK channels from GH3 cells (Shipston and Armstrong, 1996). Staurosporin (1 μM), an ATP-competitive kinase inhibitor, non-significantly reduced Po, whereas okadaic acid (100 nM), an inhibitor of serine/threonine phosphatase, significantly increased Po compared to 1 mM ATP.


Phosphorylation of BK channels modulates the sensitivity to hydrogen sulfide (H2S).

Sitdikova GF, Fuchs R, Kainz V, Weiger TM, Hermann A - Front Physiol (2014)

Mean values of BK channel open time probabilities (Popen). Three types of channels, GH3-BK (A), GH4-BK (B), and GH4-STREX (C) cells, were investigated. Bar graphs show Po-values under control conditions (left bars, white) and after extracellular application of NaHS (right bars, gray). ATP, kinase, kinase inhibitor, kinase inhibitor staurosporine (STS), or phosphatase blocker okadaic acid (OA) were added to the pipette solution; ATP0 – no ATP added; ATP1 – 1 mM ATP added; PKA (50 units); PKCsu (0.1 units/ml); PKCin – 500 nM; STS – 1 μM; OA – 100 nM. Figures are based on back transformed means and s.e.m. values (error bars), respectively. Asteriks indicate significance of H2S (300 μM NaHS equivalent to effective 34–41 μM H2S) effects according to the logit transformed data.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4228848&req=5

Figure 3: Mean values of BK channel open time probabilities (Popen). Three types of channels, GH3-BK (A), GH4-BK (B), and GH4-STREX (C) cells, were investigated. Bar graphs show Po-values under control conditions (left bars, white) and after extracellular application of NaHS (right bars, gray). ATP, kinase, kinase inhibitor, kinase inhibitor staurosporine (STS), or phosphatase blocker okadaic acid (OA) were added to the pipette solution; ATP0 – no ATP added; ATP1 – 1 mM ATP added; PKA (50 units); PKCsu (0.1 units/ml); PKCin – 500 nM; STS – 1 μM; OA – 100 nM. Figures are based on back transformed means and s.e.m. values (error bars), respectively. Asteriks indicate significance of H2S (300 μM NaHS equivalent to effective 34–41 μM H2S) effects according to the logit transformed data.
Mentions: Experimental values of BK single channel open probability (Po) for GH3, GH4, and GH4 STREX cells are listed in Table 2. Po-values are presented as mean ± standard error of mean. ATP was added to the pipette solution (intracellular membrane face). In a physiological pipette solution containing no ATP (assigned ATP0), BK single channel open probability (Po) was generally low. Addition of 1 mM ATP (ATP1) to the pipette solution most significantly increased Po (***) (Table 2 and Figure 3A). Similar findings have been reported previously from GH3 cells (Denson et al., 2001; Zhou et al., 2012), and reviewed for other cell types (Schubert and Nelson, 2001). All further solutions contained 1 mM ATP. PKA catalytic subunit (50 units/ml) altered Po, but both values were non-significant compared to ATP1 (Figure 3A), however, they were both significantly (***) different to ATP0. PKC (PKC catalytic subunit, PKCsu, 0.1 units/ml) in the pipette solution decreased BK Po non-significantly compared to ATP1, but Po was significantly different from ATP0 (*). PKC is known to cause inhibition of BK channel activity from GH cells (Shipston and Armstrong, 1996; Hall and Armstrong, 2000; Wu et al., 2007; Zhou et al., 2012, 2010). With protein kinase inhibitor pseudo-substrate (PKCin, fragment 19–31, 500 nM) Po increased non-significantly if compared to PKCsu or ATP1, but was significantly higher than during ATP0 conditions (**) if compared to ATP1. The experiments support previous results of an inhibitory role of PKC at BK channels from GH3 cells (Shipston and Armstrong, 1996). Staurosporin (1 μM), an ATP-competitive kinase inhibitor, non-significantly reduced Po, whereas okadaic acid (100 nM), an inhibitor of serine/threonine phosphatase, significantly increased Po compared to 1 mM ATP.

Bottom Line: The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution.A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology of Man and Animals, Kazan Federal University Kazan, Russia.

ABSTRACT

Introduction: Gases, such as nitric oxide (NO), carbon monoxide (CO), or hydrogen sulfide (H2S), termed gasotransmitters, play an increasingly important role in understanding of how electrical signaling of cells is modulated. H2S is well-known to act on various ion channels and receptors. In a previous study we reported that H2S increased calcium-activated potassium (BK) channel activity.

Aims: The goal of the present study is to investigate the modulatory effect of BK channel phosphorylation on the action of H2S on the channel as well as to recalculate and determine the H2S concentrations in aqueous sodium hydrogen sulfide (NaHS) solutions.

Methods: Single channel recordings of GH3, GH4, and GH4 STREX cells were used to analyze channel open probability, amplitude, and open dwell times. H2S was measured with an anion selective electrode.

Results: The concentration of H2S produced from NaHS was recalculated taking pH, temperature salinity of the perfusate, and evaporation of H2S into account. The results indicate that from a concentration of 300 μM NaHS, only 11-13%, i.e., 34-41 μM is effective as H2S in solution. GH3, GH4, and GH4 STREX cells respond differently to phosphorylation. BK channel open probability (Po) of all cells lines used was increased by H2S in ATP-containing solutions. PKA prevented the action of H2S on channel Po in GH4 and GH4 STREX, but not in GH3 cells. H2S, high significantly increased Po of all PKG pretreated cells. In the presence of PKC, which lowers channel activity, H2S increased channel Po of GH4 and GH4 STREX, but not those of GH3 cells. H2S increased open dwell times of GH3 cells in the absence of ATP significantly. A significant increase of dwell times with H2S was also observed in the presence of okadaic acid.

Conclusions: Our results suggest that phosphorylation by PKG primes the channels for H2S activation and indicate that channel phosphorylation plays an important role in the response to H2S.

No MeSH data available.


Related in: MedlinePlus