Protease-activated receptor 1-induced GABA release in cultured cortical astrocytes pretreated with GABA is mediated by the Bestrophin-1 channel

Figures & data

Figure 1. Cortical astrocytes do not contain significant amounts of GABA but can take up extracellular GABA. Representative immunocytochemistry images using anti-GABA antibody in cultured cortical astrocytes with pretreatment by GABA (left) and without GABA pretreatment (right).

Figure 2. Ca²⁺-dependent GABA release via the GABA transporter in cultured cortical astrocytes requires pretreatment by GABA. (A) Schematic illustration of sniffer-patch technique. Left pipette, pressure application of TFLLR, a selective agonist of the PAR1 receptor. Right pipette, recording pipette for HEK293T cell expressing GABA_C receptors (green). GABA released from cultured astrocyte (blue) upon TFLLR application. On completion of these experiments, GABA_C receptors of the sensor cell were fully activated by bath application of GABA (100 µM) so that the response to released GABA could be normalized according to the number of GABA receptors expressed in the sensor cell. Time-dependent reductions in sensor cell currents are due to desensitization of GABA_C receptors. (B) Microscope image of sniffer-patch assay. Ca²⁺ imaging (upper images), GABA_C receptor expressed in HEK293T cell and shRNA-transfected astrocyte (lower images). (C) Representative recordings from sniffer-patch assay for each experimental condition (left: normal medium; –GABA; right: pretreatment with GABA; +GABA). Upper trace: Ca²⁺ transient recorded from astrocyte. Lower trace: whole-cell current recorded from sensor cell (Vh = −70 mV) upon TFLLR pressure application. Diamond: TFLLR application (10 psi, 100 ms, 500 mM). (D) Summary bar graph of GABA release measured under the indicated conditions (SNAP: SNAP5114; GABA transporter inhibitor), with values normalized as described above. Significance was determined by a paired Student's t-test.

Figure 3. Ca²⁺-dependent GABA release with GABA pretreatment is mediated by Best1. (A) Representative recordings from sniffer-patch assay for each experimental condition (left: Scrambled shRNA; right: Best1 shRNA). Upper trace: Ca²⁺ transient recorded from astrocyte. Lower trace: whole-cell current recorded from sensor cell (Vh = −70 mV) upon TFLLR pressure application. Diamond: TFLLR application (10 psi, 100 ms, 500 mM). (B) Summary bar graph of GABA release measured under the conditions indicated, with values normalized as described above. Significance was determined by a paired Student's t-test.