Alzheimer's disease | mutagenesis | selectivity | neurodegeneration | polymorphism O riginally identified as a possible modifier of the age of onset of Alzheimer's disease (1, 2), calcium homeostasis modulator 1 (CALHM1) encodes a glycosylated membrane protein expressed throughout the brain that lacks homology to other proteins. Six human CALHM homologs have been identified, with alternatively spliced variants and different expression patterns throughout the body, and CALHM1 is conserved across >20 species. Expression of recombinant human CALHM1 in mammalian cells was found to strongly influence processing of amyloid precursor protein to amyloid beta (Aβ) under an experimental protocol that involved removal of Ca 2+ o for several minutes and its subsequent restoration to the bathing medium (1). This procedure resulted in a large rise of [Ca 2+ ] i . Accordingly, it was speculated that CALHM1 influences Aβ production by altering cellular Ca 2+ homeostasis. CALHM1 was found to homo-multimerize and it was speculated that it might function as an ion channel component or regulator of membrane ion conductances (1 Results CALHM1 Expression Induces a Voltage-Dependent Plasma MembraneConductance. Previously, outwardly rectified ion currents were observed in CALHM1-expressing Xenopus oocytes and CHO cells by a slow voltage ramp protocol (1). However, those experiments did not establish whether CALHM1 is an essential component of an underlying ion channel(s), accessory protein, or actual pore-forming subunit. Furthermore the detailed permeation and gating properties of the conductance were not determined. In addition, precautions were not taken to fully ensure lack of contribution of endogenous conductances. To distinguish whether CALHM1 is a unique ion channel or a regulator of endogenous channels, plasma membrane currents were recorded in Xenopus oocytes under conditions that minimized contributions of endogenous conductances (Fig. S1) (3, 4). Membrane depolarization in solutions containing 2 mM Ca 2+ and 1 mM Mg 2+ generated large outward currents that activated slowly (τ ∼ 3.11 ± 0.28 s at +60 mV; n = 10) and deactivated at hyperpolarized voltages (τ = 0.204 ± 0.022 s at −80 mV; n = 10) specifically in CALHM1-expressing oocytes (Fig. 1A). Expression of CALHM1-EGFP localized to the plasma membrane (Fig. 1B). Similar results were obtained in transiently transfected N2A mammalian neuroblastoma cells (Fig. 1C). Thus, expression of CALHM1 induced a voltage-dependent plasma membrane conductance.The monovalent ion permeabilities of this conductance were estimated by changing-bath [NaCl] in a nominally 0-Ca 2+ solution (free [Ca 2+ ] ∼10 μM) and measuring shifts of reversal potential, ΔE rev (Fig. 1D). Using the Goldman-Hodgkin-Katz (GHK) constant field equation, the relative permeabilities were estimated as P Na :P K :P Cl = 1:1.17:0.56. Similar results were obtained with bath Na + replaced by K + in either 0 or 2 mM Ca 2+ o .

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The predominant neuronal glutamate transporter, EAAC1 (for excitatory amino acid carrier-1), is localized to the dendrites and somata of many neurons. Rare presynaptic localization is restricted to GABA terminals. Because glutamate is a precursor for GABA synthesis, we hypothesized that EAAC1 may play a role in regulating GABA synthesis and, thus, could cause epilepsy in rats when inactivated. Reduced expression of EAAC1 by antisense treatment led to behavioral abnormalities, including staring-freezing episodes and electrographic (EEG) seizures. Extracellular hippocampal and thalamocortical slice recordings showed excessive excitability in antisense-treated rats. Patch-clamp recordings of miniature IPSCs (mIPSCs) conducted in CA1 pyramidal neurons in slices from EAAC1 antisense-treated animals demonstrated a significant decrease in mIPSC amplitude, indicating decreased tonic inhibition. There was a 50% loss of hippocampal GABA levels associated with knockdown of EAAC1, and newly synthesized GABA from extracellular glutamate was significantly impaired by reduction of EAAC1 expression. EAAC1 may participate in normal GABA neurosynthesis and limbic hyperexcitability, whereas epilepsy can result from a disruption of the interaction between EAAC1 and GABA metabolism.