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Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide.

Chen S, Wang J, Siegelbaum SA - J. Gen. Physiol. (2001)

Bottom Line: These results are most simply explained by the formation of heteromeric channels with novel properties.The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2.Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, ColumbiaUniversity, New York, New York 10032, USA.

ABSTRACT
Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.

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Steady-state activation curves determined in inside-out patches in the presence and absence of cAMP. (A) Average tail current activation curves for HCN1, HCN2, and coexpression of HCN1 and HCN2 in the presence (closed symbols) and absence (open symbols) of 10 μM cAMP. (left) HCN1 (7 patches); (middle) coexpression of HCN1 and HCN2 (9 patches); (right) HCN2 (10 patches). Solid lines show fit of Boltzmann relation. (B) The activation curve of Ih current generated by coexpression of HCN1 and HCN2 from a representative patch cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves obtained from A. (solid lines) HCN2, HCN1 + HCN2 (open diamonds), and HCN1 from left to right. Dashed, dotted, and dash-dotted lines: linear sums of HCN1 and HCN2 activation curves at 1:3, 1:1, 3:1 (HCN1/HCN2) ratios. (C) The average Boltzmann activation curve for Ih currents generated by coexpression of HCN1 and HCN2 (open diamonds, 7 patches) cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves. Bars indicate SEM.
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Figure 5: Steady-state activation curves determined in inside-out patches in the presence and absence of cAMP. (A) Average tail current activation curves for HCN1, HCN2, and coexpression of HCN1 and HCN2 in the presence (closed symbols) and absence (open symbols) of 10 μM cAMP. (left) HCN1 (7 patches); (middle) coexpression of HCN1 and HCN2 (9 patches); (right) HCN2 (10 patches). Solid lines show fit of Boltzmann relation. (B) The activation curve of Ih current generated by coexpression of HCN1 and HCN2 from a representative patch cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves obtained from A. (solid lines) HCN2, HCN1 + HCN2 (open diamonds), and HCN1 from left to right. Dashed, dotted, and dash-dotted lines: linear sums of HCN1 and HCN2 activation curves at 1:3, 1:1, 3:1 (HCN1/HCN2) ratios. (C) The average Boltzmann activation curve for Ih currents generated by coexpression of HCN1 and HCN2 (open diamonds, 7 patches) cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves. Bars indicate SEM.

Mentions: The effects of cAMP on the voltage dependence of gating were examined next using tail current activation curves (Fig. 5 and Table ). In the absence of cAMP, HCN1 activated at voltages that were 20 mV more positive than those required to activate HCN2 channels. For HCN1, the V1/2 of activation was −115.8 ± 1.3 mV with a slope of 6.3 ± 0.7 mV. For HCN2, the V1/2 was −135.7 ± 1.7 mV with a slope of 4.3 ± 0.3 mV. Thus, although V1/2 values were shifted by ∼50 mV relative to their values in intact oocytes, the qualitative difference in voltage dependence between HCN1 and HCN2 was maintained in the inside-out patches. In fact, the 20-mV difference in V1/2 between HCN1 and HCN2 in cell-free patches was larger than the 10-mV difference observed in intact oocytes.


Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide.

Chen S, Wang J, Siegelbaum SA - J. Gen. Physiol. (2001)

Steady-state activation curves determined in inside-out patches in the presence and absence of cAMP. (A) Average tail current activation curves for HCN1, HCN2, and coexpression of HCN1 and HCN2 in the presence (closed symbols) and absence (open symbols) of 10 μM cAMP. (left) HCN1 (7 patches); (middle) coexpression of HCN1 and HCN2 (9 patches); (right) HCN2 (10 patches). Solid lines show fit of Boltzmann relation. (B) The activation curve of Ih current generated by coexpression of HCN1 and HCN2 from a representative patch cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves obtained from A. (solid lines) HCN2, HCN1 + HCN2 (open diamonds), and HCN1 from left to right. Dashed, dotted, and dash-dotted lines: linear sums of HCN1 and HCN2 activation curves at 1:3, 1:1, 3:1 (HCN1/HCN2) ratios. (C) The average Boltzmann activation curve for Ih currents generated by coexpression of HCN1 and HCN2 (open diamonds, 7 patches) cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves. Bars indicate SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233656&req=5

Figure 5: Steady-state activation curves determined in inside-out patches in the presence and absence of cAMP. (A) Average tail current activation curves for HCN1, HCN2, and coexpression of HCN1 and HCN2 in the presence (closed symbols) and absence (open symbols) of 10 μM cAMP. (left) HCN1 (7 patches); (middle) coexpression of HCN1 and HCN2 (9 patches); (right) HCN2 (10 patches). Solid lines show fit of Boltzmann relation. (B) The activation curve of Ih current generated by coexpression of HCN1 and HCN2 from a representative patch cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves obtained from A. (solid lines) HCN2, HCN1 + HCN2 (open diamonds), and HCN1 from left to right. Dashed, dotted, and dash-dotted lines: linear sums of HCN1 and HCN2 activation curves at 1:3, 1:1, 3:1 (HCN1/HCN2) ratios. (C) The average Boltzmann activation curve for Ih currents generated by coexpression of HCN1 and HCN2 (open diamonds, 7 patches) cannot be reproduced by linear sums of average HCN1 and HCN2 activation curves. Bars indicate SEM.
Mentions: The effects of cAMP on the voltage dependence of gating were examined next using tail current activation curves (Fig. 5 and Table ). In the absence of cAMP, HCN1 activated at voltages that were 20 mV more positive than those required to activate HCN2 channels. For HCN1, the V1/2 of activation was −115.8 ± 1.3 mV with a slope of 6.3 ± 0.7 mV. For HCN2, the V1/2 was −135.7 ± 1.7 mV with a slope of 4.3 ± 0.3 mV. Thus, although V1/2 values were shifted by ∼50 mV relative to their values in intact oocytes, the qualitative difference in voltage dependence between HCN1 and HCN2 was maintained in the inside-out patches. In fact, the 20-mV difference in V1/2 between HCN1 and HCN2 in cell-free patches was larger than the 10-mV difference observed in intact oocytes.

Bottom Line: These results are most simply explained by the formation of heteromeric channels with novel properties.The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2.Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, ColumbiaUniversity, New York, New York 10032, USA.

ABSTRACT
Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.

Show MeSH
Related in: MedlinePlus