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

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.

Homocysteine augments BK channel activity and decreases exocytosis of secretory granules in rat GH3 cells

“…Ion currents were elicited in response to a series of voltage pulses from À60 to +120 mV in 20 mV steps. GH3 cells are widely used to explore the biophysical properties and modulation of BK channels activity [13][14][15][18][19][20], which are specifically inhibited by iberiotoxin (IbTx), paxilline [20,21], or tetraethylammonium (TEA) in submillimolar concentrations [22,23]. In our experiments, outward K + currents were decreased by IbTx (100 nM) or paxilline (1 lM), indicating the contribution of voltage-and Ca 2+ -dependent (BK) currents to the total outward currents (Figs 1A and 2D).…”

“…Cells were cultured at 37°C and 90% humidity in MEM (minimal essential medium) supplemented with 7% fetal calf serum and 3% horse serum [13,14]. Cells were cultured at 37°C and 90% humidity in MEM (minimal essential medium) supplemented with 7% fetal calf serum and 3% horse serum [13,14].…”

“…Cell culture GH3 pituitary cells were obtained from the European Collection of Cell cultures (ECACC). Cells were cultured at 37°C and 90% humidity in MEM (minimal essential medium) supplemented with 7% fetal calf serum and 3% horse serum [13,14]. For experiments, cells were split, grown on poly-D-lysine-coated coverslips, and used for recordings from 3 to 5 days after seeding.…”

“…Electrophysiological experiments were performed as described in detail previously [13][14][15]. In brief: recordings in the whole-cell mode or from excised patches were made with an Axopatch-200B amplifier connected to a Digidata 1322A interface, using PCLAMP10 software.…”

“…Single-channel recordings were obtained in the outsideout and inside-out mode as described previously [13][14][15]. Cell-free patches were clamped to a holding potential of +30 mV for outside-out and À30 mV for inside-out patches.…”