Characterization of genetic alterations in the cardiac HCN4 channel.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2015)
Dissertation_Stephanie Biel_Mai 2015.pdf - Accepted Version
Available under Creative Commons Attribution Non-commercial No-derivatives 3.0 de.
Download (4MB) | Preview
|Item Type:||Ph.D. Thesis|
|Title:||Characterization of genetic alterations in the cardiac HCN4 channel|
Brugada (BrS) and Sick Sinus Syndrome (SSS) are inheritable diseases, which are characterized by different cardiac arrhythmias. Tachy- and bradycardy as well as sinoatrial block, sinus rest or cardiac arrest are clinical manifestations of these diseases, which may be detected in electro¬cardio¬grams of affected persons. Both BrS and SSS are not based only on clinical, but also on symptomatic features. Symptoms like chronotropic incompetence, dizziness, syncopes, palpitations or atrial/ ventriculare fibrillation until sudden cardiac arrest are results of such cardiac arrhythmias. The appearance and occurrence of these symptoms are phenotypic and not always existing, which makes the diagnosis of these diseases based only on clinical and symptomatic criteria very difficult. Over the last years, a number of genetic abnormalities in genes encoding subunits of cardiac potassium, sodium and calcium channels, as well as in genes involved in the trafficking or regulation of these channels could be associated with both syndromes. Therefore, genetic screening of persons at a potential risk should be performed for diagnosing.
HCN4 is one of these genes, which could be associated with BrS and SSS. It encodes the hyperpolarization-activated, cyclic nucleotide-gated cation channel, which is crucial for the uninterrupted function of the sinoatrial node in the heart. HCN4 is one of four isoforms (HCN1-4) and is mainly expressed in the brain and the heart to generate a pacemaker impulse for autonomic activity. These channels are voltage-gated and are activated by the membrane hyperpolarization. In a ratio of 1:3 to 1:5, Na+ and K+ ions flow through a pore, which is formed by four subunits of the channel. As a consequence of the activity of the HCN4 channel the membrane depolarizes to the threshold voltage, which in turn triggers the subsequent action potential. Mutations in the HCN4 gene may lead to cardiac dysfunctions, which occur in diseases such as BrS and SSS. To date, more than 23 mutations in the HCN4 gene have been identified and associated with clinically established or potential sinus node dysfunctions.
In the present study, genetic screening of patients with suspected or diagnosed Brugada or Sick Sinus Syndrome was performed to identify new mutations in the HCN4 gene. In the coding HCN4 region of 62 patients, six already known and one novel sequence alteration were detected: two are located in exon 1 (N-terminus), two in exon 4 (one in the pore and one in the beginning of the C-terminus loop) and three in exon 8 (C-terminus loop). All of these six base exchanges are listed in the NCBI-database (National Center of Bio¬technology) as sequence variations with no functional modification. The new sequence variant V492F was not listed in the database and is located in the highly conserved pore region of the HCN4 channel. To prove, whether this variant is a common polymorphism or a novel mutation, 100 blood samples of healthy persons were tested. None of these samples contained this new sequence variation, what suggests a new mutation. The subsequent electrophysiological investigations on HEK293 cells expressing the HCN4-V492F mutant and three additional variants (V492A/-D/-R) at the protein position V492 indicated a reduced channel conductance in comparison to HEK293 cells expressing the HCN4-WT. This may not be exclusively attributed to a functional disorder of the channel, but may also be due to an impairment of the protein synthesis or of trafficking the channel protein to the cell surface. To address this question, the distribution of GFP tagged HCN4-WT and of mutant channels were investigated in HEK293 cells using confocal laser scanning microscopy (CLSM). The CLSM images of HEK293 cells expressing the respective mutated channels showed the same intracellular distribution and local concentration of the HCN4 protein as the images of HEK293 cells expressing the HCN4-WT. The modified technique nabopac was additionally used to isolate plasma membrane patches from cells, which had been transfected with DNA of HCN4-WT or of the several mutated channels. These highly pure plasma membrane patches showed no differences in their GFP fluorescence intensity, which indicates that an amino acid exchange in position 492 of the HCN4 protein apparently neither impaired the synthesis nor the trafficking of the channel.
Taken together, the reduced conductance of a HCN4-V492F mutant is apparently caused only by an aberrant function of the channel protein. The reduced channel conductance explains the symptoms the mutation carrier exhibits, who is suffering from unclear syncopes in resting situations and confirms the BrS diagnosis.
|Place of Publication:||Darmstadt|
|Classification DDC:||500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie|
|Divisions:||10 Department of Biology|
|Date Deposited:||05 Oct 2015 11:16|
|Last Modified:||16 Oct 2015 07:02|
|Referees:||Thiel, Prof. Dr. Gerhard and Laube, Prof. Dr. Bodo and Kauferstein, PD Dr. Silke|
|Refereed:||16 July 2015|