Electrophysiological properties of human beta-cell lines EndoC-βH1 and -βH2 conform with human beta-cells

© The Author(s) 2018Limited access to human islets has prompted the development of human beta cell models. The human beta cell lines EndoC-βH1 and EndoC-βH2 are increasingly used by the research community. However, little is known of their electrophysiological and secretory properties. Here, we monitored parameters that constitute the glucose-triggering pathway of insulin release. Both cell lines respond to glucose (6 and 20 mM) with 2- to 3-fold stimulation of insulin secretion which correlated with an elevation of [Ca2+]i, membrane depolarisation and increased action potential firing. Similar to human primary beta cells, KATP channel activity is low at 1 mM glucose and is further reduced upon increasing glucose concentration; an effect that was mimicked by the KATP channel blocker tolbutamide. The upstroke of the action potentials reflects the activation of Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. The repolarisation involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis presented a similar kinetics to human primary beta cells. The ultrastructure of these cells shows insulin vesicles composed of an electron-dense core surrounded by a thin clear halo. We conclude that the EndoC-βH1 and -βH2 cells share many features of primary human β-cells and thus represent a useful experimental model.Peer reviewedFinal Published versio

Properties of voltage-gated Na+ channels in pancreatic beta-cells: Voltage-gated Na+ channels in beta-cells

Nav channels in rodent β-cells predominantly exhibit a hyperpolarised voltage-dependence of inactivation, which is so characteristic that it can be used for functional identification of β-cells. A smaller Na+ current component (10-15%) also exists that inactivates over physiological membrane potentials and contributes to action potential firing. It has been proposed that the currents contributing to inactivation over vastly different membrane potentials reflect the contribution of different Nav α-subunits. The aim of this thesis was to investigate the contribution of individual Nav channel α-subunits to the behaviour of Nav current inactivation in β-cells and to gain insight into the underlying mechanism(s) of channel regulation. TTX-resistant variants of the Nav subunits found in β-cells (NaV1.3, NaV1.6 and NaV1.7) were used to isolate currents from individual α-subunits expressed in insulin-secreting Ins1 cells and in non-β-cells (including HEK, CHO and αTC1-6 cells). Nav1.7 inactivated at 15-20mV more negative membrane potentials than Nav1.3 and Nav1.6 in Ins1 cells. Meanwhile, all Nav subunits inactivated at ~20-30mV more negative membrane potentials in Ins1 cells than in HEK, CHO or αTC1-6 cells reflecting α-subunit and cell-specific differences in the inactivation of Nav channels. In Ins1 and primary β-cells, but never in the other cell types, widely different components of Nav inactivation (separated by 30 mV) were also observed following expression of a single type of Nav α-subunit. The more positive component exhibited a voltage-dependence of inactivation similar to that found in HEK/CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. We propose that biphasic Nav inactivation in insulin-secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition

Properties of voltage-gated Na+ channels in pancreatic beta-cells

Nav channels in rodent β-cells predominantly exhibit a hyperpolarised voltage-dependence of inactivation, which is so characteristic that it can be used for functional identification of β-cells. A smaller Na+ current component (10-15%) also exists that inactivates over physiological membrane potentials and contributes to action potential firing. It has been proposed that the currents contributing to inactivation over vastly different membrane potentials reflect the contribution of different Nav α-subunits. The aim of this thesis was to investigate the contribution of individual Nav channel α-subunits to the behaviour of Nav current inactivation in β-cells and to gain insight into the underlying mechanism(s) of channel regulation. TTX-resistant variants of the Nav subunits found in β-cells (NaV1.3, NaV1.6 and NaV1.7) were used to isolate currents from individual α-subunits expressed in insulin-secreting Ins1 cells and in non-β-cells (including HEK, CHO and αTC1-6 cells). Nav1.7 inactivated at 15-20mV more negative membrane potentials than Nav1.3 and Nav1.6 in Ins1 cells. Meanwhile, all Nav subunits inactivated at ~20-30mV more negative membrane potentials in Ins1 cells than in HEK, CHO or αTC1-6 cells reflecting α-subunit and cell-specific differences in the inactivation of Nav channels. In Ins1 and primary β-cells, but never in the other cell types, widely different components of Nav inactivation (separated by 30 mV) were also observed following expression of a single type of Nav α-subunit. The more positive component exhibited a voltage-dependence of inactivation similar to that found in HEK/CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. We propose that biphasic Nav inactivation in insulin-secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition.</p