Distribution of the Muscarinic K⁺ Channel Proteins Kir3.1 and Kir3.4 in the Ventricle, Atrium, and Sinoatrial Node of Heart

Abstract

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

Autonomic regulation of calcium and potassium channels is oppositely modulated by microtubules in cardiac myocytes

“…These ligand-gated K ϩ channels are present in ventricle at lower density than in atrium. Rat ventricular myocytes have m 2 AChR and K (ACh) channels with the former in greater abundance (5). The m 2 AChR is located primarily on the cell surface and much less so in T-tubules.…”

Agonist-induced Localization of Gq-coupled Receptors and G Protein-gated Inwardly Rectifying K+ (GIRK) Channels to Caveolae Determines Receptor Specificity of Phosphatidylinositol 4,5-Bisphosphate Signaling

“…Immunocytochemistry-Immunostaining was performed on isolated mouse atrial myocytes as described previously (20). Briefly, atrial myocytes were plated on laminin (10 g/ml)-coated coverslips for 3 h at 4°C, fixed with 4% formaldehyde in phosphate-buffered saline (PBS) on ice, permeabilized in 5% goat serum in PBS with 0.1% Triton X-100 (30 min), incubated with the primary antibody against Cav-3 and WGA-Alexa-633 (overnight), followed by Alexa Fluor 488-conjugated anti-mouse secondary antibody for 1 h. Immunofluorescence was visualized with confocal laser scanning microscopy using a LSM510 apparatus (Zeiss).…”

Comparison of formaldehyde and methanol fixatives used in the detection of ion channel proteins in isolated rat ventricular myocytes by immunofluorescence labelling and confocal microscopy

“…Myocytes were incubated with FA (a phosphate buffered 10% formalin [approximately 4% formaldehyde] solution, pH 6.8-7.2) for 30 min at room temperature as previously described [2], followed by 0.1% Triton X-100 in PBS for 30 min to permeabilise the cell membrane.…”

“…The cloning and sequencing of ion channel proteins has allowed the investigation of cardiac ion channels at the molecular level [10]. Immunolabelling of ion channels in the heart is one of the most powerful tools for understanding their localisation within myocytes [2]. Despite the importance of immunolabelling, however, ion channel proteins in cardiac myocytes are sometimes difficult to detect using the immunofluorescence labelling technique.…”