Geoffrey W. Abbott

MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia

A novel potassium channel gene has been cloned, characterized, and associated with cardiac arrhythmia. The gene encodes MinK-related peptide 1 (MiRP1), a small integral membrane subunit that assembles with HERG, a pore-forming protein, to alter its function. Unlike channels formed only with HERG, mixed complexes resemble native cardiac IKr channels in their gating, unitary conductance, regulation by potassium, and distinctive biphasic inhibition by the class III antiarrhythmic E-4031. Three missense mutations associated with long QT syndrome and ventricular fibrillation are identified in the gene for MiRP1. Mutants form channels that open slowly and close rapidly, thereby diminishing potassium currents. One variant, associated with clarithromycin-induced arrhythmia, increases channel blockade by the antibiotic. A mechanism for acquired arrhythmia is revealed: genetically based reduction in potassium currents that remains clinically silent until combined with additional stressors.

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1+ (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDRlow/C-KIT(CD117)neg population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDRlow/C-KITneg cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDRlow/C-KITneg fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.

MiRP2 Forms Potassium Channels in Skeletal Muscle with Kv3.4 and Is Associated with Periodic Paralysis

The subthreshold, voltage-gated potassium channel of skeletal muscle is shown to contain MinK-related peptide 2 (MiRP2) and the pore-forming subunit Kv3.4. MiRP2-Kv3.4 channels differ from Kv3.4 channels in unitary conductance, voltage-dependent activation, recovery from inactivation, steady-state open probability, and block by a peptide toxin. Thus, MiRP2-Kv3.4 channels set resting membrane potential (RMP) and do not produce afterhyperpolarization or cumulative inactivation to limit action potential frequency. A missense mutation is identified in the gene for MiRP2 (KCNE3) in two families with periodic paralysis and found to segregate with the disease. Mutant MiRP2-Kv3.4 complexes exhibit reduced current density and diminished capacity to set RMP. Thus, MiRP2 operates with a classical potassium channel subunit to govern skeletal muscle function and pathophysiology.

The MinK-related peptides.

Voltage-gated potassium (Kv) channels mediate rapid, selective diffusion of K+ ions through the plasma membrane, controlling cell excitability, secretion and signal transduction. KCNE genes encode a family of single transmembrane domain proteins called MinK-related peptides (MiRPs) that function as ancillary or beta subunits of Kv channels. When co-expressed in heterologous systems, MiRPs confer changes in Kv channel conductance, gating kinetics and pharmacology, and are fundamental to recapitulation of the properties of some native currents. Inherited mutations in KCNE genes are associated with diseases of cardiac and skeletal muscle, and the inner ear. This article reviews our current understanding of MiRPs--their functional roles, the mechanisms underlying their association with Kv alpha subunits, their patterns of native expression and emerging evidence of the potential roles of MiRPs in the brain. The ubiquity of MiRP expression and their promiscuous association with Kv alpha subunits suggest a prominent role for MiRPs in channel dependent systems.

The KCNE2 Potassium Channel Ancillary Subunit Is Essential for Gastric Acid Secretion

Genes in the KCNE family encode single transmembrane domain ancillary subunits that co-assemble with voltage-gated potassium (Kv) channel α subunits to alter their function. KCNE2 (also known as MiRP1) is expressed in the heart, is associated with human cardiac arrhythmia, and modulates cardiac Kv α subunits hERG and KCNQ1 in vitro. KCNE2 and KCNQ1 are also expressed in parietal cells, leading to speculation they form a native channel complex there. Here, we disrupted the murine kcne2 gene and found that kcne2 (-/-) mice have a severe gastric phenotype with profoundly reduced parietal cell proton secretion, abnormal parietal cell morphology, achlorhydria, hypergastrinemia, and striking gastric glandular hyperplasia arising from an increase in the number of non-acid secretory cells. KCNQ1 exhibited abnormal distribution in gastric glands from kcne2 (-/-) mice, with increased expression in non-acid secretory cells. Parietal cells from kcne2 (+/-) mice exhibited normal architecture but reduced proton secretion, and kcne2 (+/-) mice were hypochlorhydric, indicating a gene-dose effect and a primary defect in gastric acid secretion. These data demonstrate that KCNE2 is essential for gastric acid secretion, the first genetic evidence that a member of the KCNE gene family is required for normal gastrointestinal function.

A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs)

1. INTRODUCTION 358 1.1 Summary 358 1.2 Overview 359 1.3 Four classes of pore-forming K + channel subunits – necessary and ( sometimes ) sufficient 361 1.4 Soluble and peripheral membrane proteins that interact with P loop subunits to alter function 362 1.5 Integral membrane proteins that interact with P loop subunits to alter function 363 2. MinK DETERMINES THE FUNCTION OF MIXED CHANNEL COMPLEXES 363 2.1 The KCNE1 gene product ( MinK ) gives rise to K + -selective currents and controversy 363 2.2 MinK assembles with a P loop protein, KvLQT1, to form K + channels with unique function 364 2.2.1 Single-channel conductance of KvLQT1 and MinK/KvLQT1 channels 366 2.2.2 Other differences between KvLQT1 and MinK/KvLQT1 channels 367 2.3 MinK assembles with HERG, another P loop subunit, to regulate channel activity 368 2.4 MinK does not form chloride-selective ion channels 368 3. EXPERIMENTAL AND NATURAL MinK MUTATIONS 369 3.1 Site-directed mutations 369 3.1.1 MinK mutation alters basic channel attributes and identifies key residues 369 3.1.2 MinK is a Type I transmembrane peptide 370 3.1.3 MinK is intimately associated with the I Ks pore 370 3.1.4 The number of MinK subunits in I Ks channel complexes 372 3.2 KCNE1 mutations associated with arrhythmia and deafness alter I Ks channel function 373 3.3 Summary of MinK sites critical to I Ks channel function 374 4. MinK-RELATED PEPTIDES: AN EMERGING SUPERFAMILY 374 4.1 KCNE2, 3 and 4 encode MinK-related peptides 1, 2 and 3 ( MiRPs ) 374 4.2 MiRP1 assembles with a P loop protein, HERG, to form K + channels with unique function 375 4.2.1 MiRP1 alters activation, deactivation and single-channel conductance 376 4.2.2 MiRP1 alters regulation by K + ion and confers biphasic kinetics to channel blockade 378 4.2.3 Stable association of MiRP1 and HERG subunits 380 4.3 KCNE2 mutations are associated with arrhythmia and decreased K + flux 383 4.4 Summary of the evidence that cardiac I Kr channels are MiRP1/HERG complexes 385 5. MinK-RELATED PEPTIDES: COMMONALTIES AND IMPLICATIONS 386 5.1 Genetics and structure 386 5.2 Cell biology and function 387 6. ANSWERS, SOME OUTSTANDING ISSUES, CONCLUSIONS 387 7. ACKNOWLEDGEMENTS 389 8. REFERENCES 389 MinK and MinK-related peptide 1 (MiRP1) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K + channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRP1/HERG complexes reproduce the cardiac currents called I Ks and I Kr , respectively. Inherited mutations in KCNE1 (encoding MinK) and KCNE2 (encoding MiRP1) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily.

Kcne2 deletion uncovers its crucial role in thyroid hormone biosynthesis

Thyroid dysfunction is a global health concern, causing defects including neurodevelopmental disorders, dwarfism and cardiac arrhythmia. Here, we show that the potassium channel subunits KCNQ1 and KCNE2 form a thyroid-stimulating hormone–stimulated, constitutively active, thyrocyte K+ channel required for normal thyroid hormone biosynthesis. Targeted disruption of Kcne2 in mice impaired thyroid iodide accumulation up to eightfold, impaired maternal milk ejection, halved milk tetraiodothyronine (T4) content and halved litter size. Kcne2-deficient mice had hypothyroidism, dwarfism, alopecia, goiter and cardiac abnormalities including hypertrophy, fibrosis, and reduced fractional shortening. The alopecia, dwarfism and cardiac abnormalities were alleviated by triiodothyronine (T3) and T4 administration to pups, by supplementing dams with T4 before and after they gave birth or by feeding the pups exclusively from Kcne2+/+ dams; conversely, these symptoms were elicited in Kcne2+/+ pups by feeding exclusively from Kcne2−/− dams. These data provide a new potential therapeutic target for thyroid disorders and raise the possibility of an endocrine component to previously identified KCNE2- and KCNQ1-linked human cardiac arrhythmias.

Targeted deletion of kcne2 impairs ventricular repolarization via disruption of IK,slow1 and Ito,f

Mutations in human KCNE2, which encodes the MiRP1 potassium channel ancillary subunit, associate with long QT syndrome (LQTS), a defect in ventricular repolarization. The precise cardiac role of MiRP1 remains controversial, in part, because it has marked functional promiscuity in vitro. Here, we disrupted the murine kcne2 gene to define the role of MiRP1 in murine ventricles. kcne2 disruption prolonged ventricular action potential duration (APD), suggestive of reduced repolarization capacity. Accordingly, kcne2 (−/−) ventricles exhibited a 50% reduction in IK,slow1, generated by Kv1.5—a previously unknown partner for MiRP1. Ito,f, generated by Kv4 α subunits, was also diminished, by ~25%. Ventricular MiRP1 protein coimmunoprecipitated with native Kv1.5 and Kv4.2 but not Kv1.4 or Kv4.3. Unexpectedly, kcne2 (−/−) ventricular membrane fractions exhibited 50% less mature Kv1.5 protein than wild type, and disruption of Kv1.5 trafficking to the intercalated discs. Consistent with the reduction in ventricular K+ currents and prolonged ventricular APD, kcne2 deletion lengthened the QTc under sevoflurane anesthesia. Thus, targeted disruption of kcne2 has revealed a novel cardiac partner for MiRP1, a novel role for MiRPs in α subunit targeting in vivo, and a role for MiRP1 in murine ventricular repolarization with parallels to that proposed for the human heart.—Roepke, T. K., Kontogeorgis, A., Ovanez, C., Xu, X., Young, J. B., Purtell, K., Goldstein, P. A., Christini, D. J., Peters, N. S., Akar, F. G., Gutstein, D. E., Lerner, D. J., Abbott, G. W. Targeted deletion of kcne2 impairs ventricular repolarization via disruption of IK,slow1 and Ito,f. FASEB J. 22, 3648–3660 (2008)

Interaction of KCNE subunits with the KCNQ1 K + channel pore

KCNQ1 α subunits form functionally distinct potassium channels by coassembling with KCNE ancillary subunits MinK and MiRP2. MinK‐KCNQ1 channels generate the slowly activating, voltage‐dependent cardiac IKs current. MiRP2‐KCNQ1 channels form a constitutively active current in the colon. The structural basis for these contrasting channel properties, and the mechanisms of α subunit modulation by KCNE subunits, are not fully understood. Here, scanning mutagenesis located a tryptophan‐tolerant region at positions 338–340 within the KCNQ1 pore‐lining S6 domain, suggesting an exposed region possibly amenable to interaction with transmembrane ancillary subunits. This hypothesis was tested using concomitant mutagenesis in KCNQ1 and in the membrane‐localized ‘activation triplet’ regions of MinK and MiRP2 to identify pairs of residues that interact to control KCNQ1 activation. Three pairs of mutations exerted dramatic effects, ablating channel function or either removing or restoring control of KCNQ1 activation. The results place KCNE subunits close to the KCNQ1 pore, indicating interaction of MiRP2‐72 with KCNQ1‐338; and MinK‐59,58 with KCNQ1‐339, 340. These data are consistent either with perturbation of the S6 domain by MinK or MiRP2, dissimilar positioning of MinK and MiRP2 within the channel complex, or both. Further, the results suggest specifically that two of the interactions, MiRP2‐72/KCNQ1‐338 and MinK‐58/KCNQ1‐340, are required for the contrasting gating effects of MinK and MiRP2.

Disease-associated mutations in KCNE potassium channel subunits (MiRPs) reveal promiscuous disruption of multiple currents and conservation of mechanism

KCNE genes encode single transmembrane‐domain subunits, the MinK‐related peptides (MiRPs), which assemble with pore‐forming α subunits to establish the attributes of potassium channels in vivo. To investigate whether MinK, MiRP1, and MiRP2 operate similarly with their known native a subunit partners (KCNQ1, HERG, and Kv3.4, respectively) two conserved residues associated with human disease and influential in channel function were evaluated. As MiRPs assemble with a variety of α subunits in experimental cells and may do so in vivo, each peptide was also assessed with the other two α subunits. Inherited mutation of aspartate to asparagine (D → N) to yield D76N‐MinK is linked to cardiac arrhythmia and deafness;the analogs D82N‐MiRP1 and D90N‐MiRP2 were studied. Mutation of arginine to histidine (R → H) to yield R83H‐MiRP2 is associated with periodic paralysis; the analogs K69H‐MinK and K75H‐MiRP1 were also studied. Macroscopic and single‐channel currents showed that D → N mutations suppressed a subset of functions whereas R/K → H changes altered the activity of MinK, MiRP1, and MiRP2 with all three α subunits. The findings indicate that the KCNE peptides interact similarly with different α subunits and suggest a hypothesis: that clinical manifestations of inherited KCNE point mutations result from disruption of multiple native currents via promiscuous interactions.—Abbott, G. W., Goldstein, S. A. N. Disease‐associated mutations in KCNE potassium channel subunits (MiRPs) reveal promiscuous disruption of multiple currents and conservation of mechanism. FASEB J. 16, 390–400 (2002)