Ascorbic Acid Modulation of Calcium Channels in Pancreatic 13 Cells

We have studied the effect of ascorbic acid on voltage-dependent calcium channels in pancreatic [3 cells. Using the whole-cell and perforated-patch variants of the patch clamp technique to record calcium tail currents, we have shown that the slowly deactivating (SD) calcium channel, which is similar to the T-type channel in other cells, is inhibited in a voltage-dependent manner by ascorbic acid (AA). The other channels that carry inward current in 13 cells, FD calcium channels and sodium channels, are unaffected by AA. Ascorbic acid causes a voltagedependent decrease in the magnitude of the SD channel conductance which can be explained by the hypothesis that ~ 50-60% of the channels have their voltage dependence shifted by ~ 62 mV in the depolarizing direction. Thus, ascorbate appears to modify only a fraction of the SD channels. The activation kinetics of the ascorbate-modified channels are slower than control channels in a manner that is consistent with this hypothesis. Deactivation and inactivation kinetics are unaffected by ascorbate. These effects of ascorbate require metal ions, and it appears that some of the activity of ascorbate is due to a product of its metal catalyzed oxidation, perhaps dehydroascorbate. I N T R O D U C T I O N Voltage-dependent Ca channels play a crucial role in pancreatic [3 cell function. Ribalet and Beigelman (1980) provided the first evidence that Ca channels were involved in generating the spikes that dominate the plateau phase of glucose-induced electrical activity. The use of the patch clamp technique to directly measure Ca currents in pancreatic 13 cells was first reported by Satin and Cook (1985). Since their work, many groups have contributed to our understanding of the properties of Ca channels in 13 cells from various species, including the adult rat (Hiriart and Matteson, 1988; Sala and Matteson, 1990), neonatal rat (Satin and Cook, 1985), mouse (Rorsman and Trube, 1986; Plant, 1988), guinea pig (Rorsman and Hellman, 1988), and human (Kelly, Sutton, and Ashcroft, 1991). One aspect of the functional importance of Ca channels in 13 cells involves the suggestion that regulation of Ca channel activity by insulin secretagogues might play a role in modulating insulin secretion (Ashcroft and Rorsman, 1989). Address correspondence to Dr. Donald R. Matteson, Department of Biophysics, University of Maryland School of Medicine, 660 West Redwood St., Baltimore, Maryland 21201. j . GEN. PHYSIOL. © The Rockefeller University Press" 0022-1295/93/09/0503/21 $2.00 Volume 102 September 1993 503-523 503 on July 1, 2017 jgp.rress.org D ow nladed fom 504 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 102. 1993 The existence of multiple types of Ca channels in 13 cells has been controversial, in part because the result may be species dependent. Initially, tail current analysis was used to describe two populations of Ca channels in rat pancreatic 13 cells: slowly deactivating (SD) and fast deactivating (FD) Ca channels (Hiriart and Matteson, 1988). This conclusion was later confirmed using single channel recording techniques (Sala and Matteson, 1990; Ashcroft, Kelly, and Smith, 1990). The SD Ca channel has properties similar to the T-type channel found in other preparations (Nowycky, Fox, and Tsien, 1985): it has a small single channel conductance and it activates at relatively negative voltages, inactivates rapidly, and is blocked by low concentrations of Ni 2+ (Hiriart and Matteson, 1988). The FD channel has properties similar to the L-type Ca channel: it has a single channel conductance of ~25 pS, activates at positive voltages, and inactivates very slowly (Hiriart and Matteson, 1988; Sala and Matteson, 1990). The FD channel is required for generating the upstroke of 13 cell spikes, but a role for the SD channel has not yet been determined. Because of their relatively negative activation voltage, it is possible that SD channels contribute to the initial upstroke of the membrane potential to the plateau level. Recently, it has become evident that voltage-dependent Ca channel activity can be modulated by a variety of agents that seem to act by shifting the voltage dependence of a fraction of the channels in the depolarizing direction (Marchetti, Carbone, and Lux, 1986; Bean, 1989; Ikeda, 1991; Beech, Bernheim, and Hille, 1992). In most of these studies it was concluded that the Ca channel type that was modulated was the t0-conotoxin-sensitive N-type channel. In the experiments presented in this article, we now describe another example of this type of regulatory mechanism that involves SD (or T-type) Ca channels. Specifically, we have found that ascorbic acid shifts the voltage dependence of up to 60% of the SD Ca channels by ~ 62 mV in the depolarizing direction. Because of the known alteration of ascorbic acid metabolism that occurs in diabetes (Chatterjee and Banerjee, 1979; Som, Bosu, Mukherjee, Deb, Choudhury, Mukherjee, Chatterjee, and Chatterjee, 1981), we speculate that this SD Ca channel inhibition could contribute to a reduction in insulin secretion in diabetes.