Functional coupling of voltaje sensitive calcium channels with exocytosis of immediately releasable pool in mouse chromaffin cells”.

FERNANDO D. MARENGO

Ciudad de Buenos Aires

Simposio; 2nd Max Planck Society sponsored German-Argentine Workshop: Multiparametric Probes and Agents of Cellular Function. Simposio “Membrane associated regulatory systems”.; 2007

Max Planck Society

   Chromaffin cell exocytosis is triggered by Ca2+ entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca2+ channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31±3 fF for control conditions (ICa: 25±2 pA/pF). The N-type channel blocker -conotoxin-GVIA affected neither ICa nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker -Agatoxin-IVA decreased ICa by 37% but strongly reduced the immediately releasable pool (upper limit: 6±1 fF). We used 1A subunit knockout mice to corroborate that P/Q Ca2+ channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6±1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50mMK+) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells. 2+ entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca2+ channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31±3 fF for control conditions (ICa: 25±2 pA/pF). The N-type channel blocker -conotoxin-GVIA affected neither ICa nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker -Agatoxin-IVA decreased ICa by 37% but strongly reduced the immediately releasable pool (upper limit: 6±1 fF). We used 1A subunit knockout mice to corroborate that P/Q Ca2+ channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6±1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50mMK+) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells. Chromaffin cell exocytosis is triggered by Ca2+ entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca2+ channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31±3 fF for control conditions (ICa: 25±2 pA/pF). The N-type channel blocker -conotoxin-GVIA affected neither ICa nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker -Agatoxin-IVA decreased ICa by 37% but strongly reduced the immediately releasable pool (upper limit: 6±1 fF). We used 1A subunit knockout mice to corroborate that P/Q Ca2+ channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6±1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50mMK+) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells. 2+ entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca2+ channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31±3 fF for control conditions (ICa: 25±2 pA/pF). The N-type channel blocker -conotoxin-GVIA affected neither ICa nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker -Agatoxin-IVA decreased ICa by 37% but strongly reduced the immediately releasable pool (upper limit: 6±1 fF). We used 1A subunit knockout mice to corroborate that P/Q Ca2+ channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6±1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50mMK+) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells. Chromaffin cell exocytosis is triggered by Ca2+ entry through several voltage-dependent channel subtypes. Because it was postulated that immediately releasable vesicles are closely associated with Ca2+ channels, we wondered what channel types are specifically coupled to the release of this pool. To study this question, cultured mouse chromaffin cell exocytosis was followed by patch-clamp membrane capacitance measurements. The immediately releasable pool was estimated using paired pulse stimulation, resulting in an upper limit of 31±3 fF for control conditions (ICa: 25±2 pA/pF). The N-type channel blocker -conotoxin-GVIA affected neither ICa nor the immediately releasable pool exocytosis; although the L channel blocker nitrendipine decreased current by 50%, it did not reduce this pool significantly; and the R channel inhibitor SNX-482 significantly reduced the current but induced only a moderate decrease in the estimated IRP exocytosis. In contrast, the P/Q channel blocker -Agatoxin-IVA decreased ICa by 37% but strongly reduced the immediately releasable pool (upper limit: 6±1 fF). We used 1A subunit knockout mice to corroborate that P/Q Ca2+ channels were specifically linked to immediately releasable vesicles, and we found that also in this preparation the exocytosis of this pool was severely decreased (6±1 fF). On the other hand, application of a strong stimulus that caused the fusion of most of releasable vesicles (3 min, 50mMK+) induced similar exocytosis for wild type and knockout cells. Finally, whereas application of train stimulation on chromaffin cells derived from wild type mice provoked typical early synchronous and delayed asynchronous exocytosis components, the knockout derived cells presented a strongly depressed early exocytosis but showed a prominent delayed asynchronous component. These results demonstrate that P/Q are the dominant calcium channels associated to the release of immediately releasable pool in mouse chromaffin cells.