Ahnak is critical for cardiac Ca(V)1.2 calcium channel function and its beta-adrenergic regulation.
Defective L-type Ca2+ channel (I(CaL)) regulation is one major cause for contractile dysfunction in the heart. The I(CaL) is enhanced by sympathetic nervous stimulation: via the activation of beta-adrenergic receptors, PKA phosphorylates the alpha1C(Ca(V)1.2)- and beta2-channel subunits and ahnak, an associated 5643-amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr-ahnak on I(CaL). Binding experiments with ahnak fragments (wild-type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr-ahnak critically affected both beta2 subunit interaction and I(CaL) regulation. Binding affinity between ahnak-C1 (aa 4646-5288) and beta2 subunit decreased by approximately 50% after PKA phosphorylation or in the presence of Ile5236Thr-ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA-effects on I(CaL) increasing the amplitude by approximately 60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr-peptide and Ile5236Thr-fragment (aa 5215-5288) prevented specifically the further up-regulation of I(CaL) by isoprenaline. Hence, we suggest the ahnak-C1 domain serves as physiological brake on I(CaL). Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr-ahnak fragments to increase I(CaL). This genetic ahnak variant might cause individual differences in I(CaL) regulation upon physiological challenges or therapeutic interventions.
Haase
Hannelore
H
Max Delbrück Center for Molecular Medicine (MDC), D-13092 Berlin, Germany. haase@mdc-berlin.de
Alvarez
Julio
J
Petzhold
Daria
D
Doller
Anke
A
Behlke
Joachim
J
Erdmann
Jeanette
J
Hetzer
Roland
R
Regitz-Zagrosek
Vera
V
Vassort
Guy
G
Morano
Ingo
I
D000818:Animals; D002118:Calcium / Q000737:chemistry; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005260:Female; D005786:Gene Expression Regulation; D014644:Genetic Variation; D005982:Glutathione Transferase / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D007532:Isoleucine / Q000737:chemistry; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D008954:Models, Biological; D008957:Models, Genetic; D009154:Mutation; D020125:Mutation, Missense; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012984:Software; D013696:Temperature; D013912:Threonine / Q000737:chemistry; D014461:Ultracentrifugation; D015854:Up-Regulation
C075176:AHNAK protein, human; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010455:Peptides; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D007532:Isoleucine; D013912:Threonine; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium