A perforated patch-clamp study of calcium currents and exocytosis in chromaffin cells of wild-type and alpha(1A) knockout mice.
Simultaneous recordings of inward whole-cell Ca(2+) channel currents (I(Ca) ) and increments of capacitance as an indication of exocytosis (Delta(Cm)), were performed in voltage-clamped single adrenal chromaffin cells from wild-type and alpha(1A) subunit deficient mice, using the perforated-patch configuration of the patch-clamp technique. Using protocol #1 (one single Ca(2+) channel blocker per cell), to dissect the components of I(Ca), L channels contributed 43%, N channels 35% and P/Q channels 30% to the total I(Ca) of wild-type cells. Using protocol #2 (cumulative sequential addition of 3 microm nifedipine, 1 microm omega-conotoxin GVIA, and 1 microm omega-agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to I(Ca); an R component of around 11% remained. In wild-type mice the changes of Delta(Cm) paralleled those of I(Ca). In alpha(1A) deficient mice the L component of I(Ca) rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, Delta(Cm) associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca(2+) channel subtype, L, N, P/Q, R. Ablation of the alpha(1A) gene led to a loss of P/Q channel current and to a compensatory increase of L channel-associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in alpha(1A) -deficient mice. This may be due to altered Ca(2+) homeostasis in these mice, as compared to wild mouse chromaffin cells.
Aldea
Marcos
M
Instituto Teófilo Hernando, Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
Jun
Kisun
K
Shin
Hee-Sup
HS
Andrés-Mateos
Eva
E
Solís-Garrido
Luisa M
LM
Montiel
Carmen
C
García
Antonio G
AG
Albillos
Almudena
A
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020864:Calcium Channels, N-Type / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D020908:Calcium Channels, R-Type / Q000378:metabolism; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D025802:Electric Capacitance; D004558:Electric Stimulation; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D020867:omega-Agatoxin IVA / Q000494:pharmacology; D020866:omega-Conotoxin GVIA / Q000494:pharmacology
D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D020908:Calcium Channels, R-Type; D009543:Nifedipine; D021122:Protein Subunits; D020867:omega-Agatoxin IVA; D020866:omega-Conotoxin GVIA