Temperature: an important experimental variable in studying PKC modulation of ligand-gated ion channels.
Amphibian oocyte and mammalian heterologous expression systems are often used to investigate the function of recombinant ion channels using electrophysiological techniques. Although both systems have yielded important information, the results obtained in these systems are sometimes conflicting. Oocytes and mammalian cells differ in their physiological temperature requirements. While room temperature is within the physiological temperature range for oocytes, this temperature is far below that required by mammalian cells. Since electrophysiological studies are often performed in both oocytes and mammalian cells at room temperature, we sought to determine if recording temperature could be a factor in some disparate results obtained in these cell types. For these studies, we examined phorbol ester modulation of GABA(A) and glycine receptors. Consistent with the literature, at room temperature, PMA (phorbol 12-myristate 13-acetate) produced a large reproducible decrease in the peak amplitude of GABA and glycine-gated currents in Xenopus oocytes. In contrast, PMA was ineffective in modulating these heterologously expressed receptors at room temperature in human embryonic kidney (HEK) 293 cells. However, when electrophysiological experiments were performed at 35 degrees C in HEK 293 cells, PMA decreased the function of these receptors. Our results indicate that the temperature at which electrophysiological studies are conducted is an important experimental variable. To determine the extent to which electrophysiological recordings are performed at physiological temperatures in HEK 293 cells, a PubMed search was conducted using the search terms "patch clamp" and "HEK" for the years 2003-2004. This search revealed that only 15% of the patch clamp studies were reported to have been conducted in the temperature range of 32-37 degrees C. The results of our study indicate that temperature is an important experimental variable that requires rational consideration in the design of electrophysiological experiments.
Machu
Tina K
TK
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Forth Worth, 76203-1067, USA.
Dillon
Glenn H
GH
Huang
RenQi
R
Lovinger
David M
DM
Leidenheimer
Nancy J
NJ
D000704:Analysis of Variance; D000818:Animals; D002460:Cell Line; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D018079:Receptors, GABA / Q000502:physiology; D011985:Receptors, Serotonin / Q000502:physiology; D012701:Serotonin / Q000494:pharmacology; D013696:Temperature; D014162:Transfection / Q000379:methods; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D010703:Phorbol Esters; D018079:Receptors, GABA; D011985:Receptors, Serotonin; D012701:Serotonin; D005680:gamma-Aminobutyric Acid; D011493:Protein Kinase C; D005998:Glycine