Heat shock protein 70 (Hsp70) protects cultured motor neurons from the toxic effects of mutations in Cu/Zn-superoxide dismutase (SOD-1), which is responsible for a familial form of the disease, amyotrophic lateral sclerosis (ALS). Here, the endogenous heat shock response of motor neurons was investigated to determine whether a high threshold for activating this protective mechanism contributes to their vulnerability to stresses associated with ALS. When heat shocked, cultured motor neurons failed to express Hsp70 or transactivate a green fluorescent protein reporter gene driven by the Hsp70 promoter, although Hsp70 was induced in glial cells. No increase in Hsp70 occurred in motor neurons after exposure to excitotoxic glutamate or expression of mutant SOD-1 with a glycine--> alanine substitution at residue 93 (G93A), nor was Hsp70 increased in spinal cords of G93A SOD-1 transgenic mice or sporadic or familial ALS patients. In contrast, strong Hsp70 induction occurred in motor neurons with expression of a constitutively active form of heat shock transcription factor (HSF)-1 or when proteasome activity was sufficiently inhibited to induce accumulation of an alternative transcription factor HSF2. These results indicate that the high threshold for induction of the stress response in motor neurons stems from an impaired ability to activate the main heat shock-stress sensor, HSF1.

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K ؉ channels, the gating currents are asymmetric; although the on-gating currents are fast, the off-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between Arg-394, Glu-395, and Leu-398 on the C termini of the S4-S5 linker and Tyr-485 on the S6 of the neighboring subunit, which is responsible for the development of the slow off-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.The voltage dependence of ion channels is the basis for all electrical signaling in the central nervous system. In tetrameric voltage-gated K ϩ channels, each subunit is composed of six transmembrane ␣-helices (S1-S6), with S1-S4 forming the voltage sensing domain and S5-S6 of all four subunits forming the pore. The voltage-sensing domains are covalently connected to the S5 of the pore region by the S4-S5 linker. The intracellular gate is made up of the S6 C-terminal ends that cross each other, forming a bundle that occludes the pore when the channel is closed. Pore opening in voltage-gated K ϩ channels is controlled by the movement of the voltage sensor in which charged residues of the S4 respond to changes in membrane potential. During this conformational change, the charges are moved through the electric field, generating the transient gating currents (for review, see Ref. 1). Gating currents were first predicted by Hodgkin and Huxley and were first detected in sodium channels by Armstrong et al. (2, 3). The movement is transferred to the pore domain (electromechanical coupling) and subsequently leads to pore opening. Voltage sensor movement precedes pore opening so that the transitions the channel undergoes during electromechanical coupling are reflected in the gating currents.Activation (on) and deactivation (off) gating currents for the non-conducting Shaker-IR channel, W434F (4 -6) have been previously described (6 -8). Briefly, on-gating currents rise and decay quickly after small depolarizations but rise more slowly and exhibit more prolonged and complex decay kinetics after intermediate depolarizations and, finally, develop and decay rapidly after depolarizations large enough to activate all channels. In contrast, offgating currents, which de...

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