ACh released from parasympathetic nerves has a negative inotropic effect on ventricular as well as atrial muscle. These actions are primarily the result of the activation of the muscarinic K⁺ channel by ACh. ACh binds to the muscarinic (M₂) receptor and this leads to the activation of the muscarinic K⁺ channel via a G protein. The muscarinic K⁺ channel is a heterotetramer of Kir3.1 and Kir3.4. We have previously recorded muscarinic K⁺ current, i _K,ACh, in ventricular and atrial cells, but the density of i _K,ACh in ventricular cells was lower than in atrial cells (McMorn et al. 1993). In this study we have investigated the abundance and cellular distribution of the muscarinic K⁺ channel proteins, Kir3.1 and Kir3.4, in the atrial and ventricular myocardium by Western blotting and immunofluorescence. Rats were killed by stunning and cervical dislocation. Anti-Kir3.1 antibody against the C-terminus of Kir3.1 (aa 436-501; Alomone Labs, Jerusalem), anti-Kir3.4 antibody against the N-terminus of Kir3.4 (αCIR-N2, aa 19-32; Krapivinsky et al. 1995) and anti-M₂ receptor antibody against the i3 loop of the M₂ receptor (aa 225-359; Chemicon, Harrow) were used in this study. Western blotting showed a high abundance of Kir3.1 in atrium and a lower abundance in ventricle. Immunofluorescence and confocal microscopy on tissue sections showed high expression of Kir3.1 in atrium in the outer cell membrane and no expression in ventricle. In single atrial cells, Kir3.1 labelling was present in outer cell membrane, whereas in single ventricular cells, although there was no outer cell membrane labelling, Kir3.1 labelling was present in t-tubules. Kir3.4 labelling in atrial and ventricular tissue sections and single cells from atrium (Fig. 1A) and ventricle (Fig. 1B) was similar to that of Kir3.1 labelling. In tissue sections and single cells, M₂ receptor labelling was present in both atrium and ventricle primarily in the outer cell membrane. These results show for the first time the presence of the muscarinic K⁺ channel proteins in ventricle and confirm the functional study of McMorn et al. (1993) in that the density of the muscarinic K⁺ channel in ventricle is lower than in atrium.

Figure 1. Confocal micrographs showing Kir3.4 labelling in rat atrial (A) and ventricular (B) cells.

Krapivinsky, G., Gordon, E.A., Wickman, K. Velimirovic, B., Krapivinsky, L. & Clapham, D.E. (1995). Nature 374, 135-141.

McMorn, S.O., Harrison, S.M., Zang, W.-J., Yu, X.-J. & Boyett, M.R. (1993). Am. J. Physiol. 265, H1393-1400.