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Calcium current homeostasis and synaptic deficits in hippocampal neurons from Kelch-like 1 knockout mice.

Perissinotti PP, Ethington EA, Almazan E, Martínez-Hernández E, Kalil J, Koob MD, Piedras-Rentería ES - Front Cell Neurosci (2015)

Bottom Line: Western blot data showed the P/Q-type channel α1A subunit was less abundant in KO hippocampus compared to wildtype (WT); and P/Q-type calcium currents were smaller in KO neurons than WT during early days in vitro, although this decrease was compensated for at late stages by increases in L-type calcium current.In contrast, T-type currents did not change in culture.Synapsin I levels were also reduced in the KO hippocampus and cultured neurons displayed a concomitant reduction in synapsin I puncta and decreased miniature excitatory postsynaptic current (mEPSC) frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine Maywood, IL, USA.

ABSTRACT
Kelch-like 1 (KLHL1) is a neuronal actin-binding protein that modulates voltage-gated CaV2.1 (P/Q-type) and CaV3.2 (α1H T-type) calcium channels; KLHL1 knockdown experiments (KD) cause down-regulation of both channel types and altered synaptic properties in cultured rat hippocampal neurons (Perissinotti et al., 2014). Here, we studied the effect of ablation of KLHL1 on calcium channel function and synaptic properties in cultured hippocampal neurons from KLHL1 knockout (KO) mice. Western blot data showed the P/Q-type channel α1A subunit was less abundant in KO hippocampus compared to wildtype (WT); and P/Q-type calcium currents were smaller in KO neurons than WT during early days in vitro, although this decrease was compensated for at late stages by increases in L-type calcium current. In contrast, T-type currents did not change in culture. However, biophysical properties and western blot analysis revealed a differential contribution of T-type channel isoforms in the KO, with CaV3.2 α1H subunit being down-regulated and CaV3.1 α1G up-regulated. Synapsin I levels were also reduced in the KO hippocampus and cultured neurons displayed a concomitant reduction in synapsin I puncta and decreased miniature excitatory postsynaptic current (mEPSC) frequency. In summary, genetic ablation of the calcium channel modulator resulted in compensatory mechanisms to maintain calcium current homeostasis in hippocampal KO neurons; however, synaptic alterations resulted in a reduction of excitatory synapse number, causing an imbalance of the excitatory-inhibitory synaptic input ratio favoring inhibition.

No MeSH data available.


Related in: MedlinePlus

HVA calcium currents. (A) Current density I-V curves of HVA currents at 4–6 DIV and 8–10 DIV, VH = −50 mV. Average traces from VT = −60 to +10 mV are shown (4–6 DIV: n = 22, 18; 8–10 DIV: n = 15, 13). (B) Calcium current elicited by APW (n = 22, 34 and 21, 23); time constant values are shown to the right. *p < 0.05, Student’s t-test.
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Figure 1: HVA calcium currents. (A) Current density I-V curves of HVA currents at 4–6 DIV and 8–10 DIV, VH = −50 mV. Average traces from VT = −60 to +10 mV are shown (4–6 DIV: n = 22, 18; 8–10 DIV: n = 15, 13). (B) Calcium current elicited by APW (n = 22, 34 and 21, 23); time constant values are shown to the right. *p < 0.05, Student’s t-test.

Mentions: Results are presented as mean ± SEM. Statistical analysis was performed with the Sigma Plot 11 Software. Statistical significance was determined by P < 0.05 using Student’s t-test, unless otherwise noted. Analysis of Variance with post-hoc Duncan’s Method (ANOVA) was performed when more than 2 data sets were compared. Portions of the results reported here have been presented in abstracts at national meetings. Experimental number size (n) is always reported in order for WT and KO (n = WT, KO) in the figure legends, except for Figure 2 which is reported in the text.


Calcium current homeostasis and synaptic deficits in hippocampal neurons from Kelch-like 1 knockout mice.

Perissinotti PP, Ethington EA, Almazan E, Martínez-Hernández E, Kalil J, Koob MD, Piedras-Rentería ES - Front Cell Neurosci (2015)

HVA calcium currents. (A) Current density I-V curves of HVA currents at 4–6 DIV and 8–10 DIV, VH = −50 mV. Average traces from VT = −60 to +10 mV are shown (4–6 DIV: n = 22, 18; 8–10 DIV: n = 15, 13). (B) Calcium current elicited by APW (n = 22, 34 and 21, 23); time constant values are shown to the right. *p < 0.05, Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4285801&req=5

Figure 1: HVA calcium currents. (A) Current density I-V curves of HVA currents at 4–6 DIV and 8–10 DIV, VH = −50 mV. Average traces from VT = −60 to +10 mV are shown (4–6 DIV: n = 22, 18; 8–10 DIV: n = 15, 13). (B) Calcium current elicited by APW (n = 22, 34 and 21, 23); time constant values are shown to the right. *p < 0.05, Student’s t-test.
Mentions: Results are presented as mean ± SEM. Statistical analysis was performed with the Sigma Plot 11 Software. Statistical significance was determined by P < 0.05 using Student’s t-test, unless otherwise noted. Analysis of Variance with post-hoc Duncan’s Method (ANOVA) was performed when more than 2 data sets were compared. Portions of the results reported here have been presented in abstracts at national meetings. Experimental number size (n) is always reported in order for WT and KO (n = WT, KO) in the figure legends, except for Figure 2 which is reported in the text.

Bottom Line: Western blot data showed the P/Q-type channel α1A subunit was less abundant in KO hippocampus compared to wildtype (WT); and P/Q-type calcium currents were smaller in KO neurons than WT during early days in vitro, although this decrease was compensated for at late stages by increases in L-type calcium current.In contrast, T-type currents did not change in culture.Synapsin I levels were also reduced in the KO hippocampus and cultured neurons displayed a concomitant reduction in synapsin I puncta and decreased miniature excitatory postsynaptic current (mEPSC) frequency.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine Maywood, IL, USA.

ABSTRACT
Kelch-like 1 (KLHL1) is a neuronal actin-binding protein that modulates voltage-gated CaV2.1 (P/Q-type) and CaV3.2 (α1H T-type) calcium channels; KLHL1 knockdown experiments (KD) cause down-regulation of both channel types and altered synaptic properties in cultured rat hippocampal neurons (Perissinotti et al., 2014). Here, we studied the effect of ablation of KLHL1 on calcium channel function and synaptic properties in cultured hippocampal neurons from KLHL1 knockout (KO) mice. Western blot data showed the P/Q-type channel α1A subunit was less abundant in KO hippocampus compared to wildtype (WT); and P/Q-type calcium currents were smaller in KO neurons than WT during early days in vitro, although this decrease was compensated for at late stages by increases in L-type calcium current. In contrast, T-type currents did not change in culture. However, biophysical properties and western blot analysis revealed a differential contribution of T-type channel isoforms in the KO, with CaV3.2 α1H subunit being down-regulated and CaV3.1 α1G up-regulated. Synapsin I levels were also reduced in the KO hippocampus and cultured neurons displayed a concomitant reduction in synapsin I puncta and decreased miniature excitatory postsynaptic current (mEPSC) frequency. In summary, genetic ablation of the calcium channel modulator resulted in compensatory mechanisms to maintain calcium current homeostasis in hippocampal KO neurons; however, synaptic alterations resulted in a reduction of excitatory synapse number, causing an imbalance of the excitatory-inhibitory synaptic input ratio favoring inhibition.

No MeSH data available.


Related in: MedlinePlus