Investigating the role of Ca2+-binding site IV in barnacle troponin C

L. D. Allhouse, Q. Li, G. Guzman, T. Miller, S. Lipscomb, J. D. Potter, C. C. Ashley

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Two genetically engineered, recombinant versions of native barnacle troponin C (TnC) (BTnC2) were created from the bacterially expressed, recombinant, wild-type BTnC (BTnCWT) to investigate the role of the Ca2+- specific sites in force regulation. The mutant BTnC4- contains a single amino acid mutation in site IV which results in the inactivation of site IV Ca2+ binding; the mutant BTnCTrunc lacks the last 11 amino acids of the C- terminal, and hence most of site IV. Both mutant proteins, which retain an active site II, bind to native TnC-depleted myofibrillar bundles and restore approximately 40% of the tension-generating capacity, about half that seen with purified native BTnC1 or BTnC2. This observation implies that the Mg2+-dependent interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca2+-binding sites of BTnC2. Replacement with BTnCTrunc increases the sensitivity of the myofibrillar bundle to changes in ionic strength. Decreasing the ionic strength from 0.15 to 0.075 M increased force by 34%, a value much greater that the 8% increase seen in control bundles or bundles substituted with BTnC4-. These findings implicate TnC in determining this fibre characteristic, although this cannot be simply due to the alteration in the numbers of Ca2+ ions bound by the troponin complex since both BTnC4- and BTnCTrunc bind only 1 tool Ca2+/mol protein.

Original languageEnglish (US)
Pages (from-to)600-609
Number of pages10
JournalPflugers Archiv European Journal of Physiology
Volume439
Issue number5
DOIs
StatePublished - 2000

Keywords

  • Muscle Calcium
  • Troponin C

ASJC Scopus subject areas

  • Physiology

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