PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.

Subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ channel.

We have investigated the subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ (KATP) channel (SUR1/Kir6.2 channel) by constructing cDNA encoding a single polypeptide (beta alpha polypeptide) consisting of a SUR1 (beta) subunit and a Kir6.2 (alpha) subunit. 86Rb+ efflux and single-channel properties of COS1 cells expressing beta alpha polypeptides were similar to those of COS1 cells coexpressing alpha monomers and beta monomers. Coexpression of beta alpha polypeptides with alpha monomers inhibited the K+ currents, while coexpression with beta monomers did not. We then constructed another single polypeptide (beta alpha2) consisting of a beta subunit and a dimeric repeat of the alpha subunit. 86Rb+ efflux from COS1 cells expressing beta alpha2 polypeptides was small, but was restored by supplementation with beta monomers. These results indicate that the activity of K(ATP) channels is optimized when the alpha and beta subunits are coexpressed with a molar ratio of 1:1. Since inward rectifier K+ channels are thought to function as homo- or hetero-tetramers, this suggests that the K(ATP) channel functions as a multimeric protein, most likely a hetero-octamer composed of a tetramer of the Kir6.2 subunit and a tetramer of the SUR1 subunit.

Kir2.2v: a possible negative regulator of the inwardly rectifying K+ channel Kir2.2.

We have cloned the human genes encoding the inwardly rectifying K+ (Kir) channel subunits, Kir2.2 (hKir2.2) and its variant, termed hKir2.2v. When expressed in Xenopus oocytes, hKir2.2 produced strong inwardly rectifying K+ currents, whereas the expression of hKir2.2v did not elicit significant currents. Coexpression of hKir2.2v with hKir2.2 showed an hKir2.2v inhibition of hKir2.2 K+ currents, indicating that it acts as a negative regulator of hKir2.2 channel activity. Mutational analysis of hKir2.2v and studies of chimeras between hKir2.2 and hKir2.2v suggest that the intracellular C-terminal region of hKir2.2v participates in the negative regulation of the hKir2.2v channel activity.

Molecular diversity and functional characterization of voltage-dependent calcium channels (CACN4) expressed in pancreatic beta-cells.

Dihydropyridine-sensitive voltage-dependent calcium channels (VDCC) play a crucial role in insulin secretion. We recently have cloned a human alpha 1-subunit of the VDCC expressed in pancreatic beta-cells, designated CACN4. In this study we have isolated complementary DNAs encoding two forms of rat CACN4 (rCACN4A and rCACN4B) from a rat insulinoma RINm5F complementary DNA library. Rat CACN4A is a protein of 2203 amino acids and is the rat homolog of human CACN4, whereas rCACN4B lacks 535 amino acids in the carboxyl-terminal region, probably due to alternative splicing. We have found two additional variations, one in the intracellular loop between repeats I and II and the other in the extracellular region between the third and fourth segments of repeat IV. Reverse transcriptase-polymerase chain reaction analysis of rat pancreatic islet messenger RNA reveals that these variants are present in pancreatic islets. In addition, whole-cell voltage-clamp recordings of Chinese hamster ovary cells stably expressing the alpha 1-subunit (rCACN4A or rCACN4B) with or without the calcium channel beta 2-subunit show that coexpression of rCACN4A with the beta 2-subunit or rCACN4B with the beta 2-subunit elicits L-type VDCC currents, whereas expression of the alpha 1-subunit alone does not, indicating that CACN4 can associate functionally with the beta 2-subunit and that the beta-subunit is essential for functional expression of CACN4. These results suggest that there are various subtypes of CACN4 expressed in pancreatic beta-cells, and that both rCACN4A and rCACN4B can function as VDCC. Furthermore, the present study suggests that the expression of the beta-subunit as well as the alpha 1-subunit may participate in the regulation of insulin secretion.

Neurotrophic substance develops tetrodotoxin-sensitive action potential and increases curare-sensitivity of acetylcholine response in cultured rat myotubes.

We examined the trophic effects of a partially purified trophic substance from mouse spinal cord extract on the tetrodotoxin (TTX)-sensitivity of action potentials and on acetylcholine-sensitivity of rat skeletal myotubes in 7- and 8-day-old cultures. Many myotubes grown in control medium generate action potentials in the presence of TTX (10(-6) M). The addition of fraction E (Fr.E) from a Biogel P2 column, which exhibited trophic activity on adult denervated muscle in organ culture, decreased TTX-resistivity of action potentials of myotubes in cell culture. The trophic substance was also effective when further purified by paper chromatography and electrophoresis. The response to iontophoretically applied acetylcholine of Fr.E-treated myotubes was much more reduced by D-tubocurarine (10(-7) g/ml) than those of control cultured myotubes. No difference in morphological differentiation, protein synthesis, creatine phosphokinase activity or specific binding of [125I]alpha-bungarotoxin was observed between control and Fr.E-treated cultures. These results suggest that the trophic substance in Fr.E may be involved in the normal development of TTX-sensitive sodium channels and of acetylcholine receptor properties.