Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38.
Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.
Lee
David
D
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Ave., Newark, NJ 07103, USA.
Obukhov
Alexander G
AG
Shen
Qi
Q
Liu
Yan
Y
Dhawan
Puneet
P
Nowycky
Martha C
MC
Christakos
Sylvia
S
D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D004594:Electrophysiology; D006706:Homeostasis; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D011188:Potassium / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology
C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D064030:S100 Calcium Binding Protein G; D014700:Verapamil; D011188:Potassium; D002118:Calcium