ACTIVATION OF MUSCARINIC K+ CURRENT IN GUINEA-PIG ATRIAL MYOCYTES BY A SERUM FACTOR

1. Atrial myocytes obtained by enzymatic perfusion of hearts from adult guinea-pigs and cultured for 0-14 days were studied using the whole-cell voltage-clamp technique. 2. Superfusion of the myocytes with diluted sera (1 : 100 to 1 : 10000) from different species (human, horse, guinea-pig) evoked an inward rectifying K+ current. The voltage-dependent properties of this current were identical to those of the K+ current activated by acetylcholine (I(K(ACh))). Current density in the presence of horse serum (1 : 100) approximately corresponded to the non-desensitizing fraction of I(K(ACh)) during superfusion with 1-2 x 10(-6) m ACh. 3. During a maximal serum-evoked current, application of ACh (10(-6) m) failed to evoke additional K+ current. After switching superfusion from serum-containing to serum-free solution, the K+ current decayed 1-2 orders of magnitude slower than ACh-activated I((K(ACh)). During the decay of the serum-evoked current, a proportional increase in responsiveness to ACh was recorded. During submaximal activation of K+ current by serum, a saturating concentration of ACh resulted in a total current that was identical to the current evoked by ACh alone minus the desensitizing component. Thus, activation of K+ current by serum caused desensitization of I(K(Ach)). From these results it is concluded that sera contain a factor that activates the same population of K+ channels as ACh. 4. Irreversible activation of I(K(ACh)) by ACh in myocytes dialysed with the GTP-analogue GTP-gamma-S abolished sensitivity to serum and vice versa. 5. The effect of serum was not modified by atropine (10(-6) m) which completely blocked the response to 2 x 10(-6) m ACh. Furthermore, theophylline (1 mm), which completely inhibited I(K(ACh)) activation by adenosine (100 mum), failed to inhibit the effect of serum. Thus, neither muscarinic nor purinergic (A1) receptors are involved. 6. The peptide somatostatin (10(-6) m) and the alpha1-agonist phenylephrine (1 mum) which previously have been shown to cause activation of I(K(ACh)) channels, in the present study failed to evoke any measurable current, which excludes the involvement of the corresponding receptors. 7. Pre-incubation of the cells with pertussis toxin completely abolished I(K(ACh)) evoked by ACh. adenosine and serum. suggesting that the activating factor, like the classical agonists, causes opening of I(K(ACh)) channels via a G protein (G(i), G(K)). 8. The potency of serum to activate I(K(ACh)) was not reduced by dialysis, suggesting the molecular mass of the unknown factor to be greater-than-or-equal-to 5 kDa. No activating potency was found in the dialysing solutions. Heating samples of serum (5 min, 95-degrees-C) resulted in a complete loss of activity. 9. It is concluded that serum contains a protein which activates cardiac I(K(ACh)) channels via a hitherto unknown receptor coupled to a pertussis toxin-sensitive G protein. Preliminary attempts to further delineate the factor suggest its molecular mass to be 60-70 kDa.