Muscarinic regulation of the cardiac CFTR Cl- current by quaternary ammonium compounds.
In guinea pig ventricle, the protein kinase A-regulated Cl- current (ICl) is conducted by an alternatively spliced isoform of the cystic fibrosis transmembrane conductance regulator. We studied muscarinic regulation of this current using the whole-cell configuration of the patch-clamp technique. Acetylcholine (ACh) antagonized activation of ICl activated by 1 microM isoproterenol (ISO) in a concentration-dependent manner. The concentration of ACh that produced a half-maximal effect (K1/2) was 36 nM, the slope factor was 1.1, and the relative magnitude of the Cl- conductance at maximally effective concentrations of ACh (Gmin) was 21% of that observed in the presence of ISO alone. In the presence of 100 nM atropine, a competitive antagonist at the muscarinic receptor, the K1/2 value for ACh inhibition of ICl was increased to 4.3 microM, but the slope factor and Gmin were not affected, which indicated that the dissociation constant (KB) for atropine was < 1 nM. ACh-induced inhibition of the ISO-activated ICl was also blocked by the quaternary ammonium compound tetraethylammonium (TEA). Like atropine, TEA increased the K1/2 value for ACh inhibition of ICl without affecting the slope factor or Gmin. Schild analysis confirmed that TEA is also a competitive antagonist at the muscarinic receptor, with a KB value of 137 microM. However, tetramethylammonium (TMA), a structurally related compound, acted as an agonist at the muscarinic receptor. TMA inhibited ICl activated by 1 microM ISO with a K1/2 value of 342 microM, a slope factor of 0.87 and a Gmin value of 17%. Increasing the concentration of ISO shifted the K1/2 value for both ACh and TMA inhibition of ICl to higher concentrations and increased Gmin, without significantly affecting the slope factor. These results indicate that muscarinic regulation of ICl depends on the level of beta adrenergic stimulation in a functionally uncompetitive manner. They also suggest that TMA acts like ACh, a full agonist at the muscarinic receptor. Furthermore, we conclude that quaternary ammonium compounds, which are often used as ion substitutes and direct ion channel blockers, should be used with caution because of the significant and diverse effects they exert at muscarinic receptors.
Zakharov
S I
SI
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA.
Wagner
R A
RA
Harvey
R D
RD
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D018118:Chloride Channels / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005260:Female; D006168:Guinea Pigs; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000502:physiology*; D009206:Myocardium / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011943:Receptors, Adrenergic, beta / Q000502:physiology; D011976:Receptors, Muscarinic / Q000502:physiology*; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D018118:Chloride Channels; D008565:Membrane Proteins; D000644:Quaternary Ammonium Compounds; D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D013757:Tetraethylammonium Compounds; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D001285:Atropine; D017868:Cyclic AMP-Dependent Protein Kinases; C027917:tetramethylammonium; D007545:Isoproterenol; D000109:Acetylcholine