Neph1 regulates steady-state surface expression of Slo1 Ca2+-activated K+ channels: different effects in embryonic neurons and podocytes

Large-conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene are often components of large multiprotein complexes in excitable and nonexcitable cells. Here we show that Slo1 proteins interact with Neph1, a member of the immunoglobulin superfamily expressed in slit diaphragm domains of podocytes and in vertebrate and invertebrate nervous systems. This interaction was established by reciprocal coimmunoprecipitation of endogenous proteins from differentiated cells of a podocyte cell line, from parasympathetic neurons of the embryonic chick ciliary ganglion, and from HEK293T cells heterologously expressing both proteins. Neph1 can interact with all three extreme COOH-terminal variants of Slo1 (Slo1VEDEC, Slo1QEERL, and Slo1EMVYR) as ascertained by glutathione S-transferase (GST) pull-down assays and by coimmunoprecipitation. Neph1 is partially colocalized in intracellular compartments with endogenous Slo1 in podocytes and ciliary ganglion neurons. Coexpression in HEK293T cells of Neph1 with any of the Slo1 extreme COOH-terminal splice variants suppresses their steady-state expression on the cell surface, as assessed by cell surface biotinylation assays, confocal microscopy, and whole cell recordings. Consistent with this, small interfering RNA (siRNA) knockdown of endogenous Neph1 in embryonic day 10 ciliary ganglion neurons causes an increase in steady-state surface expression of Slo1 and an increase in whole cell Ca2+-dependent K+ current. Surprisingly, a comparable Neph1 knockdown in podocytes causes a decrease in surface expression of Slo1 and a decrease in whole cell BKCa currents. In podocytes, Neph1 siRNA also caused a decrease in nephrin, even though the Neph1 siRNA had no sequence homology with nephrin. However, we could not detect nephrin in ciliary ganglion neurons.

β1-Subunits Increase Surface Expression of a Large-Conductance Ca2+-Activated K+ Channel Isoform

Auxiliary (beta) subunits of large-conductance Ca2+-activated K+ (BKCa) channels regulate the gating properties of the functional channel complex. Here we show that an avian β1-subunit also stimulates the trafficking of BKCa channels to the plasma membrane in HEK293T cells and in a native population of developing vertebrate neurons. One C-terminal variant of BKCa α-subunits, called the VEDEC isoform after its five last residues, is largely retained in intracellular compartments when it is heterologously expressed in HEK293T cells. A closely related splice variant, called QEERL, shows high levels of constitutive trafficking to the plasma membrane. Co-expression of β1-subunits with the VEDEC isoform resulted in a large increase in surface BKCa channels as assessed by cell-surface biotinylation assays, whole cell recordings of membrane current, and confocal microscopy in HEK293T cells. Co-expression of β1-subunits slowed the gating kinetics of BKCa channels, as reported previously. Consistent with this, overexpression of β1-subunits in a native cell type that expresses intracellular VEDEC channels, embryonic day 9 chick ciliary ganglion neurons, resulted in a significant increase in macroscopic Ca2+-activated K+ current. Both the cytoplasmic N- and C-terminal domains of avian β1 are able to bind directly to VEDEC and QEERL channels. However, overexpression of the N-terminal domain by itself is sufficient to stimulate trafficking of VEDEC channels to the plasma membrane, whereas overexpression of either the cytoplasmic C-terminal domain or the extracellular loop domain did not affect surface expression of VEDEC.

Proportion of N-Type Calcium Current Activated by Action Potential Stimuli

N-type calcium currents are important in many neuronal functions, including cellular signaling, regulation of gene expression, and triggering of neurotransmitter release. Often the control of these diverse cellular functions is governed by the spatial and temporal patterns of calcium entry in subcellular compartments. Underlying this issue is the effectiveness of action potentials at triggering calcium channel opening. Chick ciliary ganglion neurons were used as model cells to study the activation of N-type calcium current during action potential depolarization. Several different action potential shapes were recorded, used as voltage command templates, and altered such that control action potential–evoked currents could be compared with those elicited by broadened action potential commands. Depending on the action potential shape used to activate calcium currents in chick ciliary ganglion neurons, and the temperature at which recordings were performed, varying proportions ( I/ Imax) of N-type calcium current could be activated. The largest proportion measured occurred using a broad ciliary ganglion cell soma action potential to activate calcium current at 37°C (100%). The smallest proportion measured occurred using a fast, high-temperature–adjusted frog motoneuron nerve terminal action potential to activate calcium current at room temperature (10%). These data are discussed with respect to the impact on cellular signaling and the regulation of transmitter release.