L-type calcium channel activity in osteoblast cells is regulated by the actin cytoskeleton independent of protein trafficking.
Voltage-dependent L-type calcium channels (VDCC) play important roles in many cellular processes. The interaction of the actin cytoskeleton with the channel in nonexcitable cells is less well understood. We performed whole-cell patch-clamp surface biotinylation and calcium imaging on different osteoblast cells to determine channel kinetics, amplitude, surface abundance, and intracellular calcium, respectively. Patch-clamp studies showed that actin polymerization by phalloidin increased the peak current density of I (Ca), whereas actin depolymerization by cytochalasin D (CD) significantly decreased the current amplitude. This result is consistent with calcium imaging, which showed that CD significantly decreased Bay K8644-induced intracellular calcium increase. Surface biotinylation studies showed that CD is not able to affect the surface expression of the pore-forming subunit α(1C). Interestingly, application of CD caused a significantly negative shift in the steady-state inactivation kinetics of I (Ca). There were decreases in the voltage at half-maximal inactivation that changed in a dose-dependent manner. CD also reduced the effect of activated vitamin D(3) (1α,25-D3) on VDCC and intracellular calcium. We conclude that in osteoblasts the actin cytoskeleton affects α(1C) by altering the channel kinetic properties, instead of changing the surface expression, and it is able to regulate 1α,25-D3 signaling through VDCC. Our study provides a new insight into calcium regulation in osteoblasts, which are essential in many physiological functions of this cell.
Department of Pharmacology, School of Pharmacy, Fudan University, Pudong New District, Shanghai, China.
Fangping
F
Li
Wenwei
W
Wang
Ming
M
Gu
Stefka
S
Gyoneva
Jie
J
Zhang
Shiying
S
Huang
Stephen F
SF
Traynelis
Hui
H
Cai
Sandra E
SE
Guggino
Xuemei
X
Zhang
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D045744:Cell Line, Tumor; D002762:Cholecalciferol / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D021381:Protein Transport / Q000187:drug effects; D051381:Rats
D020746:Calcium Channels, L-Type; D002762:Cholecalciferol; D015638:Cytochalasin D; D002118:Calcium
J Bone Miner Res. 2002 Oct;17(10):1795-800
12369783
J Biol Chem. 1994 Sep 30;269(39):23889-93
7929036
J Bone Miner Res. 2000 Jul;15(7):1275-85
10893676
Am J Physiol. 1999 Dec;277(6):C1277-83
10600780
Kidney Int. 2002 Apr;61(4):1393-406
11918746
Mol Vis. 2007 Aug 28;13:1496-507
17893673
Neuron. 2004 Apr 8;42(1):89-99
15066267
J Bone Miner Res. 1986 Oct;1(5):441-55
3332554
Cell Calcium. 2005 Nov;38(5):515-26
16137761
Am J Physiol Cell Physiol. 2009 May;296(5):C1067-78
19261907
Biol Pharm Bull. 1999 Oct;22(10):1058-63
10549855
J Bone Miner Metab. 2009;27(2):158-67
19183835
Boll Soc Ital Biol Sper. 1989 Dec;65(12):1115-8
2560636
Steroids. 2006 Apr;71(4):291-7
16457860
Endocrinology. 2003 Apr;144(4):1226-33
12639904
J Biol Chem. 2000 Mar 24;275(12):8711-8
10722713
Matrix. 1992 Dec;12(6):439-47
1287412
J Neurosci. 2008 Oct 29;28(44):11333-46
18971475
Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):4191-6
15753322
Am J Physiol Renal Physiol. 2002 Feb;282(2):F179-90
11788431
Am J Physiol Cell Physiol. 2000 Aug;279(2):C480-7
10913014
Am J Kidney Dis. 1998 Apr;31(4):729-42
9531195
Neuron. 2004 May 13;42(3):387-99
15134636
Proc Natl Acad Sci U S A. 1989 Apr;86(8):2957-60
2468165
Endocrinology. 1994 Sep;135(3):996-1003
8070395
Neuron. 1993 May;10(5):797-804
8098608
Am J Physiol Cell Physiol. 2007 May;292(5):C1830-6
17251324
J Biol Chem. 2008 Feb 15;283(7):4395-407
18048348