Ciliary Ca2+ pumps regulate intraciliary Ca2+ from the action potential and may co-localize with ciliary voltage-gated Ca2+ channels

Abstract

ABSTRACT Calcium ions (Ca2+) entering cilia through the ciliary voltage-gated calcium channels (CaV) during the action potential causes reversal of the ciliary power stroke and backward swimming in Paramecium tetraurelia. How calcium is returned to the resting level is not yet clear. Our focus is on calcium pumps as a possible mechanism. There are 23 P. tetraurelia genes for calcium pumps that are members of the family of plasma membrane Ca2+ ATPases (PMCAs). They have domains homologous to those found in mammalian PMCAs. Of the 13 pump proteins previously identified in cilia, ptPMCA2a and ptPMCA2b are most abundant in the cilia. We used RNAi to examine which PMCA might be involved in regulating intraciliary Ca2+ after the action potential. RNAi for only ptPMCA2a and ptPMCA2b causes cells to significantly prolong their backward swimming, which indicates that Ca2+ extrusion in the cilia is impaired when these PMCAs are depleted. We used immunoprecipitations (IP) to find that ptPMCA2a and ptPMCA2b are co-immunoprecipitated with the CaV channel α1 subunits that are found only in the cilia. We used iodixanol (OptiPrep) density gradients to show that ptPMCA2a and ptPMCA2b and CaV1c are found in the same density fractions. These results suggest that ptPMCA2a and ptPMCA2b are located in the proximity of ciliary CaV channels.