Isoform‐dependent modification of T‐type calcium channels by ProTxII

Abstract

The 3 isoforms of the T‐type calcium (Ca) channel are differentially expressed in the nervous system and within individual neurons (Perez‐Reyes 2003; McKay et al. 2006). There exist few experimental tools that can differentiate between channel isoforms. Here, we show that ProTxII, a highly charged peptide toxin from the venom of the tarantula Thrixopelma pruriens, differentially modifies the 3 isoforms. CaV3.1 and CaV3.3 channels are sensitive to the toxin when expressed heterologously in HEK cells. In CaV3.1, 2μM toxin produces a positive, dose‐dependent shift in the voltage range of activation of ~20mV and a reduction in macroscopic conductance of ~50%. In CaV3.3 channels 2.5μM ProTxII minimally shifted activation by ~4mV but reduced conductance by ~50%. In CaV3.2, 5μM toxin produces a minimal shift of ~5mV but no reduction in conductance. We examined the sequences of CaV3.1 and CaV3.2 for charge differences in extracellular linkers and p‐loops which may contribute to differential isoform sensitivity. We examined three reciprocal pairs in DIIS3, DII p‐loop, and DIIIS3 of these channels. Reciprocal mutations in the domain II p‐loop profoundly affected toxin sensitivity. In the CaV3.1 background the double mutation E894K/R895T resulted in reduced sensitivity to ProTxII. Conversely the K944E/T945R mutation in the CaV3.2 background conferred sensitivity. Supported by APS Summer Research Fellowship (EOH).