Abnormally Expressed Low-Voltage-Activated Calcium Channels in β-Cells From NOD Mice and a Related Clonal Cell Line

Abstract

A macroscopic low-voltage-activated (LVA) inward current was found in pancreatic beta-cells isolated from NOD mice. However, this current was not present in nondiabetic prone mouse (e.g., Swiss-Webster) pancreatic beta-cells. We performed pharmacological analyses on this current in NOD insulinoma tumor cells (NIT-1). This cell line was developed from pancreatic beta-cells of a transgenic NOD mouse. The sodium-channel blocker, tetrodotoxin (TTX; 2 micromol/l) had no effect on this LVA current. The amplitudes of currents elicited by a -20 mV test pulse retained similarity when the extracellular sodium concentration was increased from 0 to 115 mmol/l; when the extracellular calcium concentration was decreased from 10 to 2 mmol/l, there was an approximate 50% reduction of this current elicited by a -30 mV test pulse. Neither the L-type calcium-channel blocker, nifedipine (3 micromol/l), nor the N-type calcium-channel blocker, omega-CgTx-GVIA (1 micromol/l), at -30 mV produced an appreciable effect. The T-type calcium-channel blockers, nickel (3 micromol/l) and amiloride (250 micromol/l), effectively reduced the peak of this current. In 2 mmol/l calcium external solution, the threshold of voltage-dependent activation of this calcium current was approximately -65 mV, and the peak current occurred at -20 mV. Half-maximum steady-state inactivation was around -43 mV. The mean time constant of slow deactivating tail currents generated by a preceding 20 mV pulse was 2.53 ms. The intracellular free calcium concentration was two- to threefold higher in NOD mouse pancreatic beta-cells compared with Swiss-Webster pancreatic beta-cells. We concluded that there are LVA calcium channels abnormally expressed in NOD mouse beta-cells. This LVA calcium channel may be factorial to the high cytosolic free calcium concentration observed in these cells, and thereby may contribute to the pathogenesis of NOD mouse beta-cells.

Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels

“…Similar studies using the rat insulinoma cell line INS-1 found that low concentrations of nickel could completely block T-type currents (IC 50 ϭ 30 M) and partially block insulin secretion (42). Nickelsensitive T-type currents (IC 50 ϭ 3 M) have also been recorded from the mouse insulinoma cell line NIT-1 (426). Li and co-workers (463) also cloned a splice variant of Ca v 3.1 from an INS-1 cDNA library.…”

“…427). However, the I-V curve for the cytokine-induced current peaked between ϩ10 and ϩ20, while under similar recording conditions this group found that bona fide, slowly deactivating, T-type currents peaked between Ϫ20 and Ϫ10 mV (426). Human ␤-cells express both LVA and HVA currents, and in 20% of the cells tested, the LVA current was dominant (25).…”

“…25, 320) and single-channel level (17,348). Expression is species dependent, with little or no expression in rodents, but readily detectable in humans (25,426). Therefore, it is notable that LVA currents were found in ␤-cells from diabetes-prone NOD mice (426).…”

“…Expression is species dependent, with little or no expression in rodents, but readily detectable in humans (25,426). Therefore, it is notable that LVA currents were found in ␤-cells from diabetes-prone NOD mice (426). The expression of LVA currents in mouse cells has been reported to be stimulated by a 6-h treatment with cytokines (25 U/ml of interleukin-1␤ plus 300 U/ml of interferon-␥; Ref.…”

Role of Calcium in Pancreatic Islet Cell Death by IFN-γ/TNF-α

“…Because recent articles reported the expression of the LVA-Ca 2ϩ channel in islet ␤ cells from NOD mice (25) and activation of the LVA-Ca 2ϩ channel in pancreatic islet cell death by cytokines (16), we first studied whether LVA-Ca 2ϩ channel was activated in our model of pancreatic islet cell death by 100 U/ml IFN-␥ in combination with 10 ng/ml TNF-␣ (IFN-␥/TNF-␣) using MIN6N8 insulinoma cells. Under the whole-cell patch clamp condition, an inward Ca 2ϩ current appeared at above Ϫ40 mV and reached its peak at 0 mV.…”