Abstract
One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-go-go-related gene, which encodes a voltage-gated K(+) channel alpha subunit. Heterologous expression of HERG1 gives rise to K(+) currents that are similar (but not identical) to the rapid component of delayed rectification, I(Kr), in cardiac myocytes. In addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional I(Kr) channels. In the experiments here, antibodies generated against HERG1 were used to examine ERG1 protein expression in heart and in brain. In Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or RERG1 (rat ERG1) probed with antibodies targeted against the C terminus of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band is evident in blots of extracts from cells expressing MERG1b or HERG1b. In immunoblots of fractionated rat (and mouse) brain and heart membrane proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F, the 165- and 205-kDa proteins were replaced by two new bands at 175 and 130 kDa, suggesting that ERG1 is differentially glycosylated in rat/mouse brain and heart. In human heart, a single HERG1 protein with an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein (and I(Kr)) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue. Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play roles in the generation of functional cardiac I(Kr) channels.
Highlights
One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-gogo-related gene, which encodes a voltage-gated K؉ channel ␣ subunit
Specific C- and N-Anti-HERG Antibodies—The affinity purified C- and N- anti-HERG antibodies were initially tested for specificity and for potential cross reactivity with other voltagegated Kϩ channel ␣ subunits by immunohistochemistry and Western blot analysis on QT-6 cells transfected with cDNA constructs encoding HERG1, Kv1.2, Kv2.1, or Kv4.2
The 155kDa protein is identified in extracts of HERG1-transfected QT-6 cells probed with the N-anti-HERG antibody (Fig. 1B, lane c) and, as with the C-anti-HERG antibody (Fig. 1B, lane a), this band is not detected in extracts from either mock transfected QT-6 cells (Fig. 1B, lane d) or QT-6 cells transfected with the Kv1.2, Kv2.1, or Kv4.2 cDNA
Summary
One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-gogo-related gene, which encodes a voltage-gated K؉ channel ␣ subunit. N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional IKr channels. ERG1 protein (and IKr) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, play roles in the generation of functional cardiac IKr channels. ERG1 Expression in Rat, Mouse, and Human Heart has been documented in ferret heart [26], neither the expression levels nor the distributions of alternatively spliced and/or differentially processed ERG1 proteins have been examined directly to date in the mammalian myocardium
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