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

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 ananion 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, only11-13%, 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

Acetaldehyde - ethanol interactions on calcium-activated potassium (BK) channels in pituitary tumor (GH3) cells

Background: In the central nervous system ethanol (EtOH) is metabolized to acetaldehyde (ACA) primarily by the oxidative enzyme catalase. Evidence suggests that ACA is responsible for at least some of the effects on the brain that have been attributed to EtOH. Various types of ion channels which are involved in electrical signaling are targets of EtOH like maxi calcium-activated potassium (BK) channels. BK channels exhibit various functions like action potential repolarization, blood pressure regulation, hormone secretion, or transmitter release. In most neuronal and neuroendocrine preparations at physiological intracellular calcium levels, EtOH increases BK channel activity. The simultaneous presence of ACA and EtOH reflects the physiological situation after drinking and may result in synergistic as well as antagonistic actions compared to a single application of either drug. The action of ACA on electrical activity has yet not been fully established.Methods: GH3 pituitary tumor cells were used for outside-out and inside-out patch-clamp recordings of BK activity in excised patches. Unitary current amplitude, open probability and channel mean open time of BK channels were measured. Results: Extracellular EtOH raised BK channel activity. In the presence of intracellular ACA this increment of BK activity was suppressed in a dose- as well as calcium-dependent manner. Mean channel open time was significantly reduced by internal ACA, whereas BK channel amplitudes were not affected. The EtOH counteracting effect of ACA was found to depend on succession of application. EtOH was prevented from activating BK channels by pre-exposure of membrane patches to ACA. In contrast BK activation by a hypotonic solution was not affected by internal ACA. Conclusions: Our data suggest an inhibitory impact of ACA on BK activation by EtOH. ACA appears to interact specifically with EtOH at BK channels since intracellular ACA had no effect when BK channels were activated by hypotonicity