Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells
We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits o...
| Autores principales: | , , , |
|---|---|
| Formato: | JOUR |
| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12110/paper_03619230_v90_n1_p107_Weissmann |
| Aporte de: |
| id |
todo:paper_03619230_v90_n1_p107_Weissmann |
|---|---|
| record_format |
dspace |
| spelling |
todo:paper_03619230_v90_n1_p107_Weissmann2023-10-03T15:26:45Z Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells Weissmann, C. Di Guilmi, M.N. Urbano, F.J. Uchitel, O.D. CaV2.1 calcium channels Epilepsy HEK cells Neuropathic pain Pregabalin barium isoleucine pregabalin unclassified drug voltage gated calcium channel voltage gated calcium channel CaV2.1 article calcium current cellular distribution controlled study drug mechanism electrophysiology genetic transfection microscopy priority journal protein expression voltage clamp Analgesics Biophysics Calcium Channels, N-Type Electric Stimulation gamma-Aminobutyric Acid Green Fluorescent Proteins HEK293 Cells Humans Ion Channel Gating Membrane Potentials Microscopy, Confocal Patch-Clamp Techniques Transfection We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions.The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by 'whole-cell voltage-clamp' recordings, showing a maximum current peak in the I-V curve at +20mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500μM PGB, that binds α2δ subunits, for 30min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through CaV2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the CaV2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channels' currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action. © 2012 Elsevier Inc. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_03619230_v90_n1_p107_Weissmann |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
CaV2.1 calcium channels Epilepsy HEK cells Neuropathic pain Pregabalin barium isoleucine pregabalin unclassified drug voltage gated calcium channel voltage gated calcium channel CaV2.1 article calcium current cellular distribution controlled study drug mechanism electrophysiology genetic transfection microscopy priority journal protein expression voltage clamp Analgesics Biophysics Calcium Channels, N-Type Electric Stimulation gamma-Aminobutyric Acid Green Fluorescent Proteins HEK293 Cells Humans Ion Channel Gating Membrane Potentials Microscopy, Confocal Patch-Clamp Techniques Transfection |
| spellingShingle |
CaV2.1 calcium channels Epilepsy HEK cells Neuropathic pain Pregabalin barium isoleucine pregabalin unclassified drug voltage gated calcium channel voltage gated calcium channel CaV2.1 article calcium current cellular distribution controlled study drug mechanism electrophysiology genetic transfection microscopy priority journal protein expression voltage clamp Analgesics Biophysics Calcium Channels, N-Type Electric Stimulation gamma-Aminobutyric Acid Green Fluorescent Proteins HEK293 Cells Humans Ion Channel Gating Membrane Potentials Microscopy, Confocal Patch-Clamp Techniques Transfection Weissmann, C. Di Guilmi, M.N. Urbano, F.J. Uchitel, O.D. Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells |
| topic_facet |
CaV2.1 calcium channels Epilepsy HEK cells Neuropathic pain Pregabalin barium isoleucine pregabalin unclassified drug voltage gated calcium channel voltage gated calcium channel CaV2.1 article calcium current cellular distribution controlled study drug mechanism electrophysiology genetic transfection microscopy priority journal protein expression voltage clamp Analgesics Biophysics Calcium Channels, N-Type Electric Stimulation gamma-Aminobutyric Acid Green Fluorescent Proteins HEK293 Cells Humans Ion Channel Gating Membrane Potentials Microscopy, Confocal Patch-Clamp Techniques Transfection |
| description |
We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated CaV2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the CaV2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions.The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by 'whole-cell voltage-clamp' recordings, showing a maximum current peak in the I-V curve at +20mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500μM PGB, that binds α2δ subunits, for 30min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through CaV2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the CaV2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on CaV2.1 calcium channels' currents and distribution and suggest that internalization of CaV2.1 channels might be a mechanism of PGB action. © 2012 Elsevier Inc. |
| format |
JOUR |
| author |
Weissmann, C. Di Guilmi, M.N. Urbano, F.J. Uchitel, O.D. |
| author_facet |
Weissmann, C. Di Guilmi, M.N. Urbano, F.J. Uchitel, O.D. |
| author_sort |
Weissmann, C. |
| title |
Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells |
| title_short |
Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells |
| title_full |
Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells |
| title_fullStr |
Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells |
| title_full_unstemmed |
Acute effects of pregabalin on the function and cellular distribution of CaV2.1 in HEK293t cells |
| title_sort |
acute effects of pregabalin on the function and cellular distribution of cav2.1 in hek293t cells |
| url |
http://hdl.handle.net/20.500.12110/paper_03619230_v90_n1_p107_Weissmann |
| work_keys_str_mv |
AT weissmannc acuteeffectsofpregabalinonthefunctionandcellulardistributionofcav21inhek293tcells AT diguilmimn acuteeffectsofpregabalinonthefunctionandcellulardistributionofcav21inhek293tcells AT urbanofj acuteeffectsofpregabalinonthefunctionandcellulardistributionofcav21inhek293tcells AT uchitelod acuteeffectsofpregabalinonthefunctionandcellulardistributionofcav21inhek293tcells |
| _version_ |
1807318893769785344 |