The Talk-1 C-Terminus is a Charge-Sensitive Module Regulating Channel Activity

Abstract

Human beta-cell insulin secretion is dependent on Ca2+ entry through voltage-dependent Ca2+ channels (VDCCs). K+ channels modulate the beta-cell membrane potential, which influences VDCC activity. The most abundant K+ channel of the human islet is the two-pore domain K+ (K2P) channel TALK-1; however, its function is unknown. We characterized TALK-1 currents in primary mouse beta-cells and find that K2P currents are significantly reduced after genetic ablation of TALK-1. To identify mechanisms that regulate TALK-1 channel activity, we used a Tl+-sensitive fluorescent screen to assess if TALK-1 is regulated by kinases. Cells expressing TALK-1 channels were monitored for changes in Tl+ flux in response to co-expression with a constitutively active kinase (examining a total of 192 kinases), revealing 9 kinases that modify Tl+ flux through TALK-1. Interestingly, three phosphatidylinositol phosphate kinases (PIKs) including PIK3CG, PI4K2B, and PIKFYVE, increased TALK-1 channel activity. To determine if activation of TALK-1 channels by PIKs is due to build-up of PIPs, we depleted membrane PIP2 with a rapamycin-recruitable phosphatase and found that TALK-1 channel activity was reduced. As charged residues of other K+ channels mediate PIP2 sensitivity, we assessed if PIP2 influences TALK-1 channel activity through a charged residue enriched domain in the C-terminus. We found that neutralization of these charged residues significantly reduced PIP2 activation of TALK-1 channels. Importantly, PIP2 introduced via patch pipette into primary mouse beta-cells also significantly enhanced TALK-1 currents. As glucose induces Ca2+-dependent oscillations in beta-cell PIP2, we then assessed how TALK-1 regulates islet Ca2+ dynamics. We find that Ca2+ oscillation frequency is significantly increased in islets lacking TALK-1, which results in enhanced glucose-stimulated insulin secretion (GSIS). These data provide compelling evidence that TALK-1 influences GSIS by limiting excitability, and suggests a novel mechanism for phospholipid-dependent regulation of beta-cell Ca2+ entry.