KCR1, a Membrane Protein That Facilitates Functional Expression of Non-inactivating K+ Currents Associates with Rat EAG Voltage-dependent K+Channels

Abstract

Cerebellar granule neurons possess a non-inactivating K؉ current, which controls resting membrane potentials and modulates the firing rate by means of muscarinic agonists. kcr1 was cloned from the cerebellar cDNA library by suppression cloning. KCR1 is a novel protein with 12 putative transmembrane domains and enhances the functional expression of the cerebellar non-inactivating K ؉ current in Xenopus oocytes. KCR1 also accelerates the activation of rat EAG K ؉ channels expressed in Xenopus oocytes or in COS-7 cells. FarWestern blotting revealed that KCR1 and EAG proteins interacted with each other by means of their C-terminal regions. These results suggest that KCR1 is the regulatory component of non-inactivating K ؉ channels.K ϩ channels are essential components of the plasma membranes of both excitable and non-excitable cells. Three major classes have been described for voltage-gated K ϩ currents in mammalian neurons: A-type currents with rapid inactivation, delayed rectifier currents with slow inactivation, and non-inactivating outward currents (1, 2). The first two classes play an important role in shaping action potentials during repolarization and in determining the interval of an action potential. The non-inactivating outward currents can modulate firing frequency upon receptor stimulation by neurotransmitters or produce resting membrane potentials.Typical K ϩ currents that show no inactivation upon depolarization are M currents (3, 4), S currents (5), the standing outward current, I K(SO) , recently reported in cerebellar granule cells (6), and resembling currents in myoblasts (7). These currents have a common feature in that they begin to be activated from deep membrane potentials and are inhibited by neurotransmitters such as muscarinic agonists (4, 6), bradykinin (3), and serotonin (5). Except for the many important items of physiological relevance identified for such currents, molecular bases for the responsible channels are poorly understood. The cloned K ϩ channels that exhibit non-inactivating currents are the aKv5.1 (8) and ether à go-go (EAG) 1 K ϩ channels (9, 10).Recently, it has been reported that the expression of Drosophila EAG K ϩ channels in oocytes results in a slow relaxation current resembling M currents (11). However, whether a rat homologue of EAG (r-EAG) contributes to the M channel has been the subject of much debate (12, 13).To characterize low-threshold non-inactivating K ϩ currents, we measured K ϩ currents in Xenopus oocytes injected with the cerebellar poly(A) ϩ RNA, in accordance with the report by Hoger et al. (14). We realized that low-threshold non-inactivating K ϩ currents (I K(ni) ), which are similar to I K(SO) , are expressed in oocytes injected with cerebellar poly(A) ϩ RNA. To clarify the molecular information for I K(ni) , we isolated a cDNA clone required for the functional channel protein for I K(ni) by suppression cloning, which in turn is based on the capacity of antisense single strand DNA to block the expression of I K(ni) in oocytes. This strategy enabled us...

Hyperpolarization-Activated Cation Channels: From Genes to Function

“…KCR1 is a plasma membraneassociated protein with 12 putative transmembrane regions that like MiRP1 can associate with the HERG K ϩ channel (183,213). Upon overexpression in CHO cells, KCR1 reduces HCN2 current densities and affects singlechannel current parameters of this channel.…”

AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists

“…Overlay assay for mapping studies was performed as previously described 50 . Briefly, purified recombinant fusion protein (1 μg) was separated by SDS-PAGE, transferred to polyvinylidine difluoride (PVDF) or nitro-cellulose membranes, renatured by incubating at 37 °C, and incubated 1 h at room temperature with 1 μg of recombinant probes in TTBS and 5% skim milk.…”

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia

“…C29F5.4 and C07D8.6 did not reveal in vivo modulation of unc-103 (n500) in our RNAi assays. However, T24D1.4, the closest worm homologue to KCR1, which is a protein originally isolated in a screen to identify a noninactivating K ϩ current from rat cerebellum (9), did modify the activity of unc-103 (n500) in vivo. Although the function of KCR1 is not fully defined, the protein appears to be expressed in human heart and influences HERG sensitivity to drug blockage in heterologous expression systems and transfected cardiac myocytes (10).…”