Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels.
A calmodulin (CaM) binding 'IQ' domain on the L-type Ca(2+) channel (LTCC) C terminus and calmodulin kinase II (CaMK) both signal increases in LTCC opening probability (P(o)) by shifting LTCCs into a gating mode (mode 2) with long openings through a process called facilitation. However, the mechanism whereby CaMK and the IQ domain are targeted to LTCCs is unknown. Endogenous CaMK is targeted to LTCCs in excised cell membrane patches because LTCC P(o) increased significantly in CaM-enriched (20 microM) bath solution and this effect was prevented by a specific CaMK inhibitory peptide, but not by an inactive control peptide. Pre-exposure of myocytes to the cytoskeletal disrupting agents nocodazole (microtubule specific) or cytochalasin D (microfilament specific) prevented the effects of CaM-dependent increases in P(o) of LTCCs in excised membrane patches. Neither cytochalasin D nor nocodazole altered the distribution of LTCC gating modes under basal conditions in on-cell mode or excised cell membrane patches, but each of these agents occluded the response of LTCCs to exogenous, constitutively active CaMK and to an IQ-mimetic peptide (IQmp). Cytochalasin D and nocodazole pretreatment also prevented LTCC facilitation that followed a cell membrane depolarizing prepulse. In contrast, cytochalasin D and nocodazole did not affect the increase in LTCC P(o) or prevent the shift to mode 2 gating in response to protein kinase A, indicating that cytoskeletal disruption specifically prevents prepulse, CaMK and IQ-dependent LTCC facilitation.
Dzhura
Igor
I
Department of Internal Medicine, Vanderbilt University Medical Center, Vanderbilt University, Nashville, TN 37232-6300, USA.
Wu
Yuejin
Y
Colbran
Roger J
RJ
Corbin
Jackie D
JD
Balser
Jeffrey R
JR
Anderson
Mark E
ME
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000187:drug effects*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects*; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D015739:Nocodazole / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011817:Rabbits
D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D010455:Peptides; D015638:Cytochalasin D; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015739:Nocodazole