title: Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.
abstract: Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.
authors.affiliation: Laboratorio de Electrofisiologia, Instituto de Cardiologia y Cirugia Cardiovascular, Havana, Cuba.
authors.forename: Julio L
authors.initials: JL
authors.lastname: Alvarez
authors.forename: Daria
authors.initials: D
authors.lastname: Petzhold
authors.forename: Ines
authors.initials: I
authors.lastname: Pankonien
authors.forename: Joachim
authors.initials: J
authors.lastname: Behlke
authors.forename: Michiyoshi
authors.initials: M
authors.lastname: Kouno
authors.forename: Guy
authors.initials: G
authors.lastname: Vassort
authors.forename: Ingo
authors.initials: I
authors.lastname: Morano
authors.forename: Hannelore
authors.initials: H
authors.lastname: Haase
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar
chemical_list: C489572:Ahnak protein, mouse; C489573:Ahnak protein, rat; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D009363:Neoplasm Proteins