Metabolic stress shifts KCa3.1 channel function from an important physiological regulator of membrane potential to a proapoptotic factor

The calcium activated potassium channel KCa3.1 is known to influence the course of several diseases like sickle cell disease and diabetic kidney disease or the progression of certain forms of cancer. In pancreatic beta cells the KCa3.1 channel influences phases of electrical activity and therefore insulin secretion. Recently it was shown that inhibition of KCa3.1 channels ameliorates glucose tolerance in a diabetic mouse model by influencing inflammatory pathways, but the mode of action remains unclear [1]. We further elucidated how KCa3.1 channels influence metabolically stressed beta cells.

Islets or islet cells were isolated from C57BL/6N mice and cultured for 48 h in the presence of glucolipotoxicity (25 mM glucose and 100 µM palmitate) or under control conditions. After this period the KCa3.1 expression was determined by immunofluorescence and several electrophysiological parameters were characterized by patch clamp and microelectrode experiments. Cell death was determined by a TUNEL assay and mitochondrial KCa3.1 channels were analysed by Western Blot (MIN-6 cells).

KCa3.1 channels were upregulated by metabolic stress as already reported [1]. KCa3.1 current density was almost doubled in metabolically stressed cells compared to control cells at a physiological membrane potential (senicapoc sensitive current: 5 vs. 11 pA/pF at 10 mV, n=28-31). Furthermore, glucolipotoxicity decreased the dependency of KCa3.1 channel activation on calcium originated from influx through L-type calcium channels. Reduced coupling of these two channels might be the reason for the lowered fraction of current through KCa3.1 channels contributing to the Kslow current. The Kslow current is composed of current through KCa3.1 and KATP channels and determines electrical bursts. The Kslow current was measured in control cells and metabolically stressed cells and the fraction, which was blocked by the KCa3.1 inhibitor senicapoc (1 µM) was calculated (44±20 vs. 22±16 %, n=12, p <0.01). In agreement with this observation, islets of Langerhans were hyperexcitable after culture in glucolipotoxic medium and senicapoc lost its stimulatory effect (percentage of time with electrical activity in control islets: 7±3 vs. 24±13 %, n=9, p<0.001; metabolically stressed islets: 80±15 vs. 83±15 %, n=10, n.s.). Interestingly, treatment with glucolipotoxicity elevates a potassium current activated by NMDA receptors that contain the subunit GluN2B (current blocked by the GluN2B antagonist Ro25-6981: 10 vs. 21 pA/pF at 10 mV, n=30-32). This suggests an increased coupling of KCa channels with these NMDA receptors. This hypothesis was verified by the observation that senicapoc does not reduce potassium currents in metabolically stressed cells in the presence of the GluN2B antagonist Ro 25-6981 (senicapoc-sensitive current in the absence and presence of Ro 25-6981: 6.9 vs. -2.6 pA/pF at 10 mV, n=30) while opposing results were acquired in control cells (senicapoc-sensitive current in the absence and presence of Ro25-6981: 6.9 vs. 4.3 pA/pF at 10 mV, n=30-31). Finally, it was shown that inhibition of KCa3.1 channels by senicapoc (1 µM) or maurotoxin (20 nM) protects from apoptosis (rate of apoptosis, percentage of glucolipotoxic challenged cells: 36±15 / 46±25 %, n=6, p<0.01). It is known that mitochondrial KCa3.1 channels influence cell survival in different types of cancer cells. We gained first evidence that KCa3.1 channels are expressed in mitochondria of the murine beta cell line MIN6. Whether mitoKCa3.1 channels are involved in the regulation of islet cell apoptosis remains to be investigated.

In conclusion, KCa3.1 channels are upregulated by metabolic stress in pancreatic beta cells but the impact of the channel on cell physiology is shifted from an important positive modulator of electrical activity to a proapoptotic factor. These changes seem to be caused by an alteration in ion channel coupling. The proapoptotic properties of KCa3.1 channels could be one pathway of NMDA receptor overactivation induced harm by metabolic stress to pancreatic beta cells.