The Neuronal β4 Subunit Increases the Unitary Conductance of L-type Voltage-gated Calcium Channels in PC12 Cells

Jessica M Schjött,Shu-Chan Hsu,Mark R Plummer

doi:10.1074/jbc.m302059200

Jessica M Schjött, Shu-Chan Hsu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m302059200

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Abstract

beta subunits of voltage-gated calcium channels influence channel behavior in numerous ways, including enhancing the targeting of alpha1 subunits to the plasma membrane and shifting the voltage dependence of activation and inactivation. Of the four beta subunits that have been identified, beta 4 is of particular interest because mutation of its alpha1 subunit interaction domain produces severe neurological defects. Its differential distribution in the hippocampus prompted us to examine whether this subunit was responsible for the heterogeneity of hippocampal L-type calcium channels. To study the functional effects of the beta 4 subunit on native L-type calcium channels, we transfected beta 4 cDNA subcloned out of embryonic hippocampal neurons into PC12 cells, a cell line that contains the beta 1, beta 2, and beta 3 subunits but not the beta 4 subunit. Cell-attached single-channel recordings of L-type channel activity from untransfected and transfected PC12 cells compared with recordings obtained from hippocampal neurons revealed an effect of the beta 4 subunit on single-channel conductance. L-type channels in untransfected PC12 cells had a significantly smaller conductance (19.8 picosiemens (pS)) than L-type channels in hippocampal neurons (22 pS). After transfection of beta 4, however, L-type single-channel conductance was indistinguishable between the two cell types. Our data suggest that calcium channel beta 4 subunits affect the conductance of L-type calcium channels and that native hippocampal L-type channels contain the beta 4 subunit.

Highlights

Voltage-gated calcium channels are multisubunit proteins that show considerable heterogeneity in neuronal and nonneuronal cell types
We have shown previously that cultured hippocampal neurons possess two kinds of L-type voltage-gated calcium channels, Lp and Ls, that differ in their response to depolarizing voltage pulses [12, 13] and high frequency stimulation [16]
Cell-attached single-channel recordings from undifferentiated PC12 cells made with identical procedures revealed L-type channel activity that was highly reminiscent of Ls channels in hippocampal neurons (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Primary Hippocampal Cell Cultures—Hippocampal neurons were obtained from embryonic day 18 Sprague-Dawley rats. Cultures were maintained at 37 °C in a 95% air/5% CO2 humidified incubator in serum-free medium made up of MEM and Ham’s F12 (1:1) and containing 6 mg/ml glucose, 100 ␮g/ml transferrin, 25 ␮g/ml insulin, 20 nM progesterone, 60 ␮M putrescine, 30 nM selenium, and 0.5 units/ml and 0.5 mg/ml of penicillin and streptomycin, respectively. Cell-attached single-channel recordings were obtained using standard techniques [27] in a solution containing (in mM): 140 potassium gluconate, 10 HEPES-KOH, and 5 EGTA. PC12 Cell Culture—PC12 cells were grown in culture flasks and maintained at 37 °C in a 95% air/5% CO2 humidified incubator in Dulbecco’s modified Eagle’s medium with 7.5% fetal bovine serum, 7.5% horse serum, and 1 ml penicillin/Streptomycin (10,000 units/ml and 10 mg/ml, respectively).

RESULTS

DISCUSSION

With regard to the composition of native hippocampal Lp and