Cytoplasmic and Extracellular IsK Peptides Activate Endogenous K and Cl Channels in Xenopus Oocytes

Abstract

The last few years have been an exciting time for our understanding of the molecular structure and diversity of voltagegated K ϩ channels belonging to the Shaker-like superfamily (for review, see Refs. 1-5). Contrastingly, since its original cloning by expression in Xenopus oocytes, the nature of the IsK protein (or minK) remained a mystery (for review, see Refs. 6 and 7). From a structural point of view, IsK is an exception to the family of K ϩ channels. IsK is a 14.5-kDa type III glycoprotein with one transmembrane segment which has no sequence homology with other cloned functional channels (8). It is a member of a family of small bitopic membrane proteins which induce upon expression in Xenopus oocytes slowly activating voltage-dependent currents (6,7,9). This family includes phospholemman (10), influenza virus M2 protein (11), CHIF (12), and Mat-8 (13). When expressed in Xenopus oocytes or in HEK 293 cells, the IsK protein evokes a unique slowly activating, voltage-dependent K ϩ -selective current that closely resembles the slow component I Ks of the cardiac delayed rectifier (8,14). Two main hypotheses concerning the nature of this protein were raised. The first was that IsK alone is sufficient to form a voltage-gated K ϩ channel, because mutations in the transmembrane domain altered the gating of the K ϩ current expressed in Xenopus oocytes and changed the relative permeabilities of NH 4 ϩ and Cs ϩ versus K ϩ (15, 16). However, attempts to express IsK as a K ϩ channel in a variety of host cells and after lipid bilayer reconstitution have failed (17). The second hypothesis was that IsK forms functional K ϩ channels by association with an endogenous oocyte factor or with pre-existing silent channels (18 -20). Furthermore, IsK mutagenesis suggested that the amino-terminal domain is critical for the induction of Cl Ϫ currents while the carboxyl-terminal domain is critical for the activation of K ϩ channel activity (18). These findings hinted at the possibility that IsK is a regulatory subunit of heteromultimeric channel complexes rather than a channel per se.To test this hypothesis, synthetic IsK hydrophilic peptides were applied to untreated Xenopus oocytes. In agreement with IsK membrane topology, we show here that internal or external application of IsK peptides derived from the carboxyl-and amino-terminal domains are sufficient to activate slow K ϩ or Cl Ϫ currents, respectively. The peptide-activated channels displayed characteristics similar to those exhibited by the native IsK protein, namely voltage dependence, activation kinetics, ion selectivity, and pharmacology. Our data provide clear evidence for the nature of IsK as a prototypic member of a family of short membrane transport proteins with regulatory function. EXPERIMENTAL PROCEDURESPeptide Synthesis and Purification-Peptides were synthesized by a solid-phase method on pyridine-2-aldoximine methyl-amino acid resin (0.15 meq) (21), as described previously (22). The peptides were subjected to amino acid analysis to confirm their composition.Electrop...

KvLQT1, a voltage-gated potassium channel responsible for human cardiac arrhythmias

“…Because ion channels can be modulated through their interaction with other subunits (20), an interesting possibility is that KvLQT1 associates with the minK polypeptide to form the channel responsible for the I Ks current. MinK, the gene believed to encode cardiac I Ks (11,21,22), was a logical candidate given the debate as to whether minK encodes a Kϩ channel as a homomultimer, whether it associates with a separate ancillary protein or subunit, or whether minK merely regulates the activity of another channel protein (23). To test for the interaction, KVLQT1 cRNA was coinjected with minK cRNA into Xenopus oocytes and membrane currents were recorded (Fig.…”

KCNE2 is colocalized with KCNQ1 and KCNE1 in cardiac myocytes and may function as a negative modulator of IKs current amplitude in the heart

“…In our oocyte experiments, KCNE1 or KCNE2 cRNA injection is delayed by 1 to 2 days after KCNQ1 cRNA injection. Indeed, injecting a purified KCNE1 protein 32 or even just a KCNE1 fragment 33 into the cytoplasm of oocytes expressing KCNQ1 can induce an I Ks -like current, suggesting that the newly introduced KCNE1 or KCNE1 fragment can associate with preformed KCNQ1 channels in the cell membrane and modulate their function. This scenario, in conjunction with the possibility that the KCNQ1 channels' KCNE-binding pockets can accommodate multiple and mixed KCNE subunits, sets the stage for dynamic regulation of I Ks current amplitude in cardiac myocytes that coexpress KCNQ1, KCNE1, and KCNE2.…”

Mink Subdomains That Mediate Modulation of and Association with Kvlqt1

“…Two naturally occurring point mutations in the COOH terminus have been linked to congenital long QT syndrome, suggesting that this subdomain is functionally critical (Splawski et al, 1997). Furthermore, Ben-Efraim et al (1996) injected synthetic peptide encoding a fragment of the MinK COOH terminus (amino acid residues 67-93) into Xenopus oocytes and recorded currents resembling I Ks , presumably by modulating an endogenous KvLQT1. To determine whether the COOH terminus is necessary for modulation, we coexpressed a COOH-terminus deleted (MinK ⌬Cterm) MinK construct (deletion of residues 67-129) and human KvLQT1 in Xenopus oocytes.…”