Atsushi Inanobe

Clustering and enhanced activity of an inwardly rectifying potassium channel, Kir4.1, by an anchoring protein, PSD-95/SAP90.

An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/KAB-2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line. When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the Ser-Asn-Val motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95 compared with cells expressing Kir4.1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1. Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore, PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.

Immunolocalization of an inwardly rectifying K+ channel, KAB‐2 (Kir4.1), in the basolateral membrane of renal distal tubular epithelia

Immunolocalization of KAB−2 (Kir4.1), an inwardly ectifying K+ channel with a putative ATP‐binding domain, was examined in rat kidney where expression of KAB−2 mRNA was previously shown. Anti‐KAB−2 antibody was raised in rabbit and then affinity‐purified. An immunohistochemical study revealed that KAB‐2 immunoreactivity was detected specifically in the basolateral membrane of distal tubular epithelia. Therefore, KAB‐2 is the first K+ channel shown to be localized in the basolateral membrane of renal epithelia. The finding suggests that KAB‐2 may contribute to supplying K+ to the Na+‐K+ pump, which is abundant in the basolateral membrane of distal tubular epithelia, as well as to maintenance of the deep negative membrane potential of these cells.

Secretagogue-induced Exocytosis Recruits G Protein-gated K+ Channels to Plasma Membrane in Endocrine Cells

Stimulation-regulated fusion of vesicles to the plasma membrane is an essential step for hormone secretion but may also serve for the recruitment of functional proteins to the plasma membrane. While studying the distribution of G protein-gated K+ (KG) channels in the anterior pituitary lobe, we found KG channel subunits Kir3.1 and Kir3.4 localized on the membranes of intracellular dense core vesicles that contained thyrotropin. Stimulation of these thyrotroph cells with thyrotropin-releasing hormone provoked fusion of vesicles to the plasma membrane, increased expression of Kir3.1 and Kir3.4 subunits in the plasma membrane, and markedly enhanced KG currents stimulated by dopamine and somatostatin. These data indicate a novel mechanism for the rapid insertion of functional ion channels into the plasma membrane, which could form a new type of negative feedback control loop for hormone secretion in the endocrine system.

Acetylcholine and adenosine activate the G protein-gated muscarinic K+ channel in ferret ventricular myocytes

The properties of the K+ channel activated by acetylcholine (ACh) and adenosine (Ado) were examined in single ferret ventricular myocytes using patch-clamp techniques. In the whole-cell configuration, ACh and Ado induced an inwardly rectifying K+ current and shortened the action potential duration. The effect of ACh was blocked by atropine, while the Ado effect was interrupted by 8-cyclopenty1,1,2-dipropyl xanthine, a specific Ado A1 receptor antagonist. In cell-attached recordings, ACh and Ado added to the pipette solution activated a single population of inwardly rectifying K+ channels, distinct from the iK1 channel. The channel had a slope conductance of ∼ 40 pS in symmetrical 150 mM K+ solutions and a mean open time of 0.8 ms. Excision of the patch into the inside-out patch configuration in guanosine triphosphate (GTP)-free solution abolished the channel activity. The channel was reversibly reactivated by adding GTP to the intracellular side of the patch. GTPγS activated the channel irreversibly. When the inside-out patch was treated with the A protomer of pertussis toxin (PTX), intracellular GTP no longer activated the K+ channel. The results show that ferret ventricular myocytes possess a K+ channel activated by both muscarinic and Ado A1 receptors. Its electrophysiological properties and the gating by a PTX-sensitive G protein in a membrane-delimited fashion are identical with those of the muscarinic K+ channels in nodal and atrial tissues of other species. In conclusion, the G protein-gated muscarinic K+ channel is expressed in ferret ventricular myocardium and may underlie the direct negative inotropism of ACh and Ado in this tissue.

G Protein-Gated K+ Channels

Upon stimulation of vagal nerves, “Vagusstoff,” which was afterwards identified as acetylcholine (ACh), is released from the axonal terminals of the vagal nerve and decelerates the heart beat. This historical discovery by OTTOLOEWI in the 1920s, established the concept of synaptic chemical transmission (LOEWI 1921; LOEwI and NAVARATIL 1926). Since then, many physiologists have been trying to elucidate the mechanisms underlying ACh-induced bradycardia. DEL-CASTILLO and KATZ (1955) described hyperpolarization of the membrane induced by ACh in frog heart. HUTTER and TRAUTWEIN (1955) measured an increase of the K+ efflux across the cardiac cell membrane under vagal stimulation. TRAUTWEIN and DUDEL (1958) showed an increase of K+ conductance under the voltage clamp condition. TRAUTWEIN and his colleagues (NOMA and TRAUTWEIN 1978; OSTERRIEDER et al. 1981) further analyzed the relaxation kinetics of the ACh-induced K+ current in the rabbit sinoatrial node and proposed that ACh induces activation of a specific population of K+ channels, named muscarinic K+ (KACN) channels, to decelerate the pacemaker activity. The single channel currents of the KACN channels were recorded for the first time by Sakmann et al. (1983), who showed that the channel exhibits an inwardly rectifying property but gating kinetics different from that of the background inwardly rectifying K+ (IK1) channel in cardiac myocytes. In 1985-6, it was discovered that pertussis toxin (PTX)-sensitive heterotrimeric G proteins are involved in the activation of the KACN channel by m2-muscarinic and A1-,purinergic receptors (PFAFFINGER et al. 1985; BREITWIESER and SZABO 1985; KURACHI et al. 1986a,b,c). Because the KACh channel could be activated by intracellular GTP (in the presence of agonists) and GTPγS (even in the absence of agonists) in cell-free inside-out patches (KURACHI et al. 1986a,b,c), the system is delimited to the cell membrane, leading to the proposal that the channel is directly activated by G protein subunits. The G protein responsible for activation of KACh channels was designated GK according to its function(BREITWIESER and SZABO 1985).