Reverse engineering the L-type Ca2+ channel α1c subunit in adult cardiac myocytes using novel adenoviral vectors

Anand N. Ganesan, Brian O'Rourke, Christoph Maack, Henry Colecraft, Agnieszka Sidor, David C. Johns

Research output: Contribution to journalArticle

Abstract

The α1c subunit of the cardiac L-type Ca2+ channel, which contains the channel pore, voltage- and Ca2+-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca2+ channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length α1c gene cassettes into the adenovirus backbone. Wild-type (α1c-wt) and mutant (α1c-D-) Ca2+ channel adenoviruses were constructed. The α1c-D- contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both α1c-wt and α1c-D- expressed robustly in A549 cells (peak L-type Ca2+ current (ICaL) at 0 mV: α1c-wt -9.94 ± 1.00 pA/pF, n = 9; α 1c-D- -10.30 pA/pF, n = 12). ICaL carried by α1c-D- was markedly less sensitive to nitrendipine (IC 50 17.1 μM) than α1c-wt (IC50 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 μM nitrendipine blocked only 51 ± 5% (n = 9) of ICaL in α1c-D--expressing myocytes, in comparison to 86 ± 8% (n = 9) of ICaL in control myocytes. Moreover, in 20 μM nitrendipine, calcium transients could still be evoked in α1c-D--transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca2+ release. These α1c adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca2+ channel regulation in the native myocyte background.

Original languageEnglish (US)
Pages (from-to)749-754
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume329
Issue number2
DOIs
StatePublished - Apr 8 2005

Keywords

  • Adenovirus
  • Dihydropyridine
  • Excitation-contraction coupling
  • Gene transfer
  • Ion channel

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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