Michael Koenen

Location of a threonine residue in the α−subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel

By the combination of cDNA manipulation and functional analysis of normal and mutant acetylcholine receptor (AChR) channels of Torpedo expressed in Xenopus laevis oocytes determinants of ion flow were localized in the bends bordering the putative M2 transmembrane segment (Imoto et al. 1988). We now report that in the rat muscle AChR, substitution of a threonine residue in the α-subunit localized in the M2 transmembrane segment increases or decreases the channel conductance, depending on the size of the amino acid side chain located at this position. This threonine residue (αT264) is located adjacent to the cluster of charged amino acids that form the intermediate anionic ring (Imoto et al. 1988). This effect is pronounced for the large alkali cations Cs+, Rb+, K+ whereas for Na+ the effect is much smaller. Taken together the results suggest that the threonine residues at position 264 in the two α-subunits together with the amino acids of the intermediate anionic ring form part of a narrow region close to the cytoplasmic mouth of the AChR channel.

A general and rapid mutagenesis method using polymerase chain reaction.

The construction of deletions, insertions and point mutations in DNA sequences is a powerful approach to analysing the function and structure of genes and their products. Here, we present a fast and efficient method using the polymerase chain reaction to introduce mutations into cDNAs coding for the alpha-, gamma- and epsilon-subunit of the rat muscle acetylcholine receptor. Two flanking primers and one mutant oligo, in conjunction with supercoiled plasmid DNA and a fragment of the target DNA are sufficient to introduce the mutation by two PCR amplifications. Our method permits directing the location of mutations anywhere in the target gene with a very low misincorporation rate, as no substitution could be detected within 9600 bp. The utility of this approach is demonstrated by the rapid introduction and analysis of eleven mutations into three different cDNAs. Any kind of mutation can be introduced with an efficiency of at least 50%.

Different mechanisms regulate muscle-specific AChR gamma- and epsilon-subunit gene expression.

Five different subunits, alpha, beta, gamma, delta and epsilon, constitute the acetylcholine receptors from mammalian skeletal muscle. Their corresponding mRNA levels are regulated differentially. In particular, mRNAs encoding the gamma‐ and epsilon‐subunits, which specify two AChR isoforms, show a reciprocal behaviour during synapse formation and maturation. We have isolated 5′ flanking sequences of the gamma‐ and epsilon‐subunit genes that confer muscle‐specific expression upon transient transfection of primary cultures of rat muscle cells. The gamma‐subunit gene fragment contains two adjacent CANNTG sequence motifs that are essential for muscle‐specific transcriptional activity suggesting transactivation by helix‐loop‐helix proteins. The epsilon‐subunit gene fragment carries only a single CANNTG consensus motif which is not required for expression in transfected muscle cells. This sequence motif is, however, necessary to repress transcriptional activity in non‐muscle cells and thus may control the muscle‐specific expression of the epsilon‐subunit gene. The results suggest that CANNTG motifs together with their 3′ and 5′ flanking nucleotides provide binding sites for both activating as well as repressing trans‐acting factors. These elements could thus contribute to the muscle‐specific expression of AChR subunit genes.

Asymmetry of the Rat Acetylcholine Receptor Subunits in the Narrow Region of the Pore

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the α-subunit revealed that a threonine residue (αT264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the β-, γ-, and δ-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the α-, β-, or γ-subunits, but it is smaller if the valine is located in the δ-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of α-, γ- and δ-subunits, where the δ-subunit serine is a main determinant of the channel conductance.

Genetic suppression of atrial fibrillation using a dominant-negative ether-a-go-go–related gene mutant

BACKGROUND Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Gene therapy-dependent modulation of atrial electrophysiology may provide a more specific alternative to pharmacological and ablative treatment strategies. OBJECTIVE We hypothesized that genetic inactivation of atrial repolarizing ether-a-go-go-related gene (ERG) K(+) currents using a dominant-negative mutant would provide rhythm control in AF. METHODS Ten domestic swine underwent pacemaker implantation and were subjected to atrial burst pacing to induce persistent AF. Animals were then randomized to receive either AdCERG-G627S to suppress ERG/I(Kr) currents or green fluorescent protein (AdGFP) as control. Adenoviruses were applied using a novel hybrid technique combining atrial virus injection and epicardial electroporation to increase transgene expression. RESULTS In pigs treated with AdCERG-G627S, the onset of persistent AF was prevented (n = 2) or significantly delayed compared with AdGFP controls (12 ± 2.1 vs. 6.2 ± 1.3 days; P < .001) during 14-day follow-up. Effective refractory periods were prolonged in the AdCERG-G627S group compared with AdGFP animals (221.5 ± 4.7 ms vs. 197.0 ± 4.7 ms; P < .006). Impairment of left ventricular ejection fraction (LVEF) during AF was prevented by AdCERG-G627S application (LVEF(CERG-G627S) = 62.1% ± 4.0% vs. LVEF(GFP) = 30.3% ± 9.1%; P < .001). CONCLUSION Inhibition of ERG function using atrial AdCERG-G627S gene transfer suppresses or delays the onset of persistent AF by prolongation of atrial refractoriness in a porcine model. Targeted gene therapy represents an alternative to pharmacological or ablative treatment of AF.

cAMP Sensitivity of HCN Pacemaker Channels Determines Basal Heart Rate But Is Not Critical for Autonomic Rate Control

Background—HCN channels activate the pacemaker current If, which is thought to contribute significantly to generation and regulation of heart rhythm. HCN4 represents the dominant isotype in the sinoatrial node and binding of cAMP was suggested to be necessary for autonomic heart rate regulation. Methods and Results—In a candidate gene approach, a heterozygous insertion of 13 nucleotides in exon 6 of the HCN4 gene leading to a truncated cyclic nucleotide-binding domain was identified in a 45-year-old woman with sinus bradycardia. Biophysical properties determined by whole-cell patch-clamp recording of HEK293 cells demonstrated that mutant subunits (HCN4-695X) were insensitive to cAMP. Heteromeric channels composed of wild-type and mutant subunits failed to respond to cAMP-like homomeric mutant channels, indicating a dominant-negative suppression of cAMP-induced channel activation by mutant subunits. Pedigree analysis identified 7 additional living carriers showing similar clinical phenotypes, that is, sinus node dysfunction with mean resting heart rate of 45.9±4.6 bpm (n=8) compared with 66.5±9.1 bpm of unaffected relatives (n=6; P<0.01). Clinical evaluation revealed no ischemic or structural heart disease in any family member. Importantly, mutant carriers exhibited normal heart rate variance and full ability to accelerate heart rate under physical activity or pharmacological stimulation. Moreover, mutant carriers displayed distinctive sinus arrhythmias and premature beats linked to adrenergic stress. Conclusions—In humans, cAMP responsiveness of If determines basal heart rate but is not critical for maximum heart rate, heart rate variability, or chronotropic competence. Furthermore, cAMP-activated If may stabilize heart rhythm during chronotropic response.

Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (α2βγδ) and adult types of AChR (α2βεδ). Mice lacking the ε‐subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal‐type AChR with an adult‐type AChR preserves myoblast fusion, muscle and end‐plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end‐plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.

Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors.

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the gamma‐subunit of the fetal muscle acetylcholine receptor (gamma‐AChR) is replaced by the epsilon‐subunit in the adult muscle receptor (epsilon‐AChR), was investigated by analysing the structure of gamma‐ and epsilon‐subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single‐channel conductance. 2. The gamma‐ and epsilon‐subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilon(gamma)‐ or gamma(epsilon)‐subunits were constructed from the parental epsilon‐ and gamma‐subunits, respectively. Exchanging the four hydrophobic segments (M1‐M4) of the gamma‐subunit for those of the epsilon‐subunit and vice versa completely reversed the difference in conductance between gamma‐AChR and epsilon‐AChR channels. 4. Effects of single‐ and multiple‐point mutations in M1‐M4 segments of gamma‐ and epsilon‐subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (gamma‐subunit) for serine/isoleucine (epsilon‐subunit) in M2, and the lysine (gamma‐subunit) for glutamine (epsilon‐subunit) exchanges in the regions flanking the M2 segment.

Organisation of the murine 5‐HT3 receptor gene and assignment tohuman chromosome 11

We have isolated the murine gene encoding the 5‐HT3 receptor (5‐HT3R), a member of the ligand‐gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5‐HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use ofexon 9 splice acceptor sites generated two 5‐HT3R variants. The 5‐HT3R gene, whose structure is closely related to neuronal and muscle AChRα genes, as demonstrated by four common splice junctions, was localised on human chromosome 11.

Transcription profiling of HCN-channel isotypes throughout mouse cardiac development

Hyperpolarization-activated ion channels, encoded by four mammalian genes (HCN1-4), contribute in an important way to the cardiac pacemaker current If. Here, we describe the transcription profiles of the four HCN genes, the NRSF, KCNE2 and Kir2.1 genes from embryonic stage E9.5 dpc to postnatal day 120 in the mouse. Embryonic atrium and ventricle revealed abundant HCN4 transcription but other HCN transcripts were almost absent. Towards birth, HCN4 was downregulated in the atrium and almost vanished from the ventricle. After birth, however, HCN isotype transcription changed remarkably, showing increased levels of HCN1, HCN2 and HCN4 in the atrium and of HCN2 and HCN4 in the ventricle. HCN3 showed highest transcription at early embryonic stages and was hardly detectable thereafter. At postnatal day 10, HCN4 was highest in the sinoatrial node, being twofold higher than HCN1 and fivefold higher than HCN2. In the atrium, HCN4 was similar to HCN1 and sevenfold higher than HCN2. In the ventricle, in contrast, HCN2 was sixfold higher than HCN4, while HCN1 was absent. Subsequently all HCN isotype transcripts declined to lower adult levels, while ratios of HCN isotypes remained stable. In conclusion, substantial changes of HCN isotype transcription throughout cardiac development suggest that a regulated pattern of HCN isotypes is required to establish and ensure a stable heart rhythm. Furthermore, constantly low HCN transcription in adult myocardium may be required to prevent atrial and ventricular arrhythmogenesis.