Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

73-POS Board 73 Ca 2+ Permeability of Bacterial Sodium Channel Heterotetramers Can Explain the EEEE

Paradox in Voltage-gated Na + and Ca 2+ Channels. Zeyu Zheng , Olena Fedorenko, Stephen Roberts. Lancaster University, Lancaster, United Kingdom.

The highly selective permeation of ions through voltage gated Na + and Ca 2+ channels can be explained in terms of fixed negative charged in the pore region ( Q f ). However, the Q f associated with bacterial voltage-gated Na + channels (bacNa v s) is reported to be equivalent to that found in mammalian Ca 2+ selective channels (i.e. a pore region with an “EEEE motif”; Q f = -4). This anomaly could be explained if we consider the conserved aspartate residue located in domain II (D 2 p 51 ) of L-type Ca 2+ channels and defining the pore with an “EEEED motif” (i.e. Q f = -5). To investigate this hypothesis, we generated concatemers of bacNa v s to allow the asymmetrical mutation of the selectivity filter (SF) and enable 1 e step changes in the Q f value associated with the SF. Western blot analysis, immunofluorescence microscopy and patch clamp were employed to evaluate the functionality of these concatenated bacNa v s and test the hypothesis that a Q f value of at least -5 is necessary for Ca 2+ permeation. This approach was corroborated with the use of defined mixtures of cDNAs encoding for NaChBac mutants exhibiting different Q f values. Preliminary patch clamp investigations suggest that a | Q f | ≥ 5 is necessary for Ca 2+ permeation, consistent with the hypothesis that Ca 2+ permeation in L-type Ca 2+ channels is mediated by a EEEED locus. The work was supported by EPSRC (grant No.\ EP/M015831/1).

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