Rostislav Bychkov

Characterization of an apamin-sensitive small-conductance Ca2+-activated K+ channel in porcine coronary artery endothelium: relevance to EDHF

The apamin‐sensitive small‐conductance Ca2+‐activated K+ channel (SKCa) was characterized in porcine coronary arteries. In intact arteries, 100 nM substance P and 600 μM 1‐ethyl‐2‐benzimidazolinone (1‐EBIO) produced endothelial cell hyperpolarizations (27.8±0.8 mV and 24.1±1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1‐EBIO response but substance P continued to induce a hyperpolarization (25.8±0.3 mV). In freshly‐isolated endothelial cells, outside‐out patch recordings revealed a unitary K+ conductance of 6.8±0.04 pS. The open‐probability was increased by Ca2+ and reduced by apamin (100 nM). Substance P activated an outward current under whole‐cell perforated‐patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7±0.03 pS inhibited by d‐tubocurarine was observed infrequently. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SKCa was detected by RT – PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri‐nuclear regions of both endothelial and smooth muscle cells. In conclusion, the porcine coronary artery endothelium expresses an apamin‐sensitive SKCa containing the SK3 subunit. These channels are likely to confer all or part of the apamin‐sensitive component of the endothelium‐derived hyperpolarizing factor (EDHF) response.

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF

This study characterizes the K+ channel(s) underlying charybdotoxin‐sensitive hyperpolarization of porcine coronary artery endothelium. Two forms of current‐voltage (I/V) relationship were evident in whole‐cell patch‐clamp recordings of freshly‐isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. Single‐channel, outside‐out patch recordings revealed a 17.1±0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca2+ demonstrated that the open probability was increased by Ca2+. This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1±0.7 mV) were reduced by apamin (100 nM; 17.0±1.8 mV) whereas those to 1‐ethyl‐2‐benzimidazolinone (1‐EBIO, 600 μM, 21.0±0.3 mV) were unaffected (21.7±0.8 mV). Substance P, bradykinin (100 nM) and 1‐EBIO evoked charybdotoxin‐sensitive, iberiotoxin‐insensitive whole‐cell perforated‐patch currents. A porcine homologue of the intermediate‐conductance Ca2+‐activated K+ channel (IK1) was identified in endothelial cells. In conclusion, porcine coronary artery endothelial cells express an intermediate‐conductance Ca2+‐activated K+ channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin‐sensitive component of the EDHF response.

A delayed ATP-elicited K+ current in freshly isolated smooth muscle cells from mouse aorta.

Adenosine 5′‐triphosphate (ATP) activated two sequential responses in freshly isolated mouse aortic smooth muscle cells. In the first phase, ATP activated Ca2+‐dependent K+ or Cl− currents and the second phase was the activation of a delayed outward current with a reversal potential of −75.9±1.4 mV. A high concentration of extracellular K+ (130 mM) shifted the reversal potential of the delayed ATP‐elicited current to −3.5±1.3 mV. The known K+‐channel blockers, iberiotoxin, charybdotoxin, glibenclamide, apamin, 4‐aminopyridine, Ba2+ and tetraethylammonium chloride all failed to inhibit the delayed ATP‐elicited K+ current. Removal of ATP did not decrease the amplitude of the ATP‐elicited current back to the control values. The simultaneous recording of cytosolic free Ca2+ and membrane currents revealed that the first phase of the ATP‐elicited response is associated with an increase in intracellular Ca2+, while the second delayed phase develops after the return of cytosolic free Ca2+ to control levels. ATP did not activate Ca2+‐dependent K+ currents, but did elicit Ca2+‐independent K+ currents, in cells dialyzed with ethylene glycol‐bis (2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA). The delay of activation of Ca2+‐independent currents decreased from 10.5+3.4 to 1.27±0.33 min in the cells dialyzed with 2 mM EGTA. Adenosine alone failed to elicit a Ca2+‐independent K+ current but simultaneous application of ATP and adenosine activated the delayed K+ current. Intracellular dialysis of cells with guanosine 5′‐O‐(2‐thiodiphosphate) transformed the Ca2+‐independent ATP‐elicited response from a sustained to a transient one. A phospholipase C inhibitor, U73122 (1 μM), was shown to abolish the delayed ATP‐elicited response. These results indicate that the second phase of the ATP‐elicited response was a delayed Ca2+‐independent K+ current activated by exogenous ATP. This phase might represent a new vasoregulatory pathway in vascular smooth muscle cells.

Purinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aorta

Aim:  Exogenous ATP elicits a delayed calcium‐independent K+ current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current.

Protein Kinase A and C Regulate Leak Potassium Currents in Freshly Isolated Vascular Myocytes from the Aorta

We tested the hypothesis that protein kinase A (PKA) inhibits K2P currents activated by protein kinase C (PKC) in freshly isolated aortic myocytes. PDBu, the PKC agonist, applied extracellularly, increased the amplitude of the K2P currents in the presence of the “cocktail” of K+ channel blockers. Gö 6976 significantly reduced the increase of the K2P currents by PDBu suggesting the involvement of either α or β isoenzymes of PKC. We found that forskolin, or membrane permeable cAMP, did not inhibit K2P currents activated by the PKC. However, when PKA agonists were added prior to PDBu, they produced a strong decrease in the K2P current amplitudes activated by PKC. Inhibition of PDBu-elicited K2P currents by cAMP agonists was not prevented by the treatment of vascular smooth muscle cells with PKA antagonists (H-89 and Rp-cAMPs). Zn2+ and Hg2+ inhibited K2P currents in one population of cells, produced biphasic responses in another population, and increased the amplitude of the PDBu-elicited K+ currents in a third population of myocytes, suggesting expression of several K2P channel types. We found that cAMP agonists inhibited biphasic responses and increase of amplitude of the PDBu-elicited K2P currents produced by Zn2+ and Hg2. 6-Bnz-cAMp produced a significantly altered pH sensitivity of PDBu-elicited K2P-currents, suggesting the inhibition of alkaline-activated K2P-currents. These results indicate that 6-Bnz-cAMP and other cAMP analogs may inhibit K2P currents through a PKA-independent mechanism. cAMP analogs may interact with unidentified proteins involved in K2P channel regulation. This novel cellular mechanism could provide insights into the interplay between PKC and PKA pathways that regulate vascular tone.